Use of alpha-glucosidase inhibitors to treat alphavirus infections

ABSTRACT

The present invention provides methods for treating a flavivirus infection, including hepatitis C virus (HCV) infection, in an individual suffering from a flavivirus infection. In some embodiments, the methods involve administering to an individual in need thereof an effective amount of an agent that inhibits enzymatic activity of a membrane-bound α-glucosidase inhibitor. In other embodiments, the methods involve administering to an individual in need thereof effective amounts of an α-glucosidase inhibitor and at least one additional therapeutic agent.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Patent App. Nos.60/660.074. filed Mar. 8, 2005 and 60/707,891, filed Aug. 12, 2005, aswell as U.S. Non-provisional application Ser. No. 11/370.535, filed Mar.7, 2006, which applications arc incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

This invention is in the field of treatment of alphavirus infection.

BACKGROUND OF THE INVENTION

The family Alphaviridae includes influenza viruses, parainfluenzaviruses, picornaviruses, polio virus, flaviviruses, e.g. yellow fevervirus, the four serotypes of dengue virus, Japanese encephalitis virus,Tick-borne encephalitis virus, West Nile virus, hepatitis viruses, andmany other disease causing viruses.

Hepatitis C virus is an illustrative example of the family ofalphaviruses. Hepatitis C virus (HCV) infection is the most commonchronic blood borne infection in the United States. Although the numbersof new infections have declined, the burden of chronic infection issubstantial, with Centers for Disease Control estimates of 3.9 million(1.8%) infected persons in the United States. Chronic liver disease isthe tenth leading cause of death among adults in the United States, andaccounts for approximately 25,000 deaths annually, or approximately 1%of all deaths. Studies indicate that 40% of chronic liver disease isHCV-related, resulting in an estimated 8,000-10,000 deaths each year.HCV-associated end-stage liver disease is the most frequent indicationfor liver transplantation among adults.

Antiviral therapy of chronic hepatitis C has evolved rapidly over thelast decade, with significant improvements seen in the efficacy oftreatment. Nevertheless, even with combination therapy using pegylatedIFN-α plus ribavirin, 40% to 50% of patients fail therapy, i.e., arenonresponders or relapsers. These patients currently have no effectivetherapeutic alternative. In particular, patients who have advancedfibrosis or cirrhosis on liver biopsy are at significant risk ofdeveloping complications of advanced liver disease, including ascites,jaundice, variceal bleeding, encephalopathy, and progressive liverfailure, as well as a markedly increased risk of hepatocellularcarcinoma.

The high prevalence of chronic HCV infection has important public healthimplications for the future burden of chronic liver disease in theUnited States. Data derived from the National Health and NutritionExamination Survey (NHANES III) indicate that a large increase in therate of new HCV infections occurred from the late 1960s to the early1980s, particularly among persons between 20 to 40 years of age. It isestimated that the number of persons with long-standing HCV infection of20 years or longer could more than quadruple from 1990 to 2015, from750,000 to over 3 million. The proportional increase in persons infectedfor 30 or 40 years would be even greater. Since the risk of HCV-relatedchronic liver disease is related to the duration of infection, with therisk of cirrhosis progressively increasing for persons infected forlonger than 20 years, this will result in a substantial increase incirrhosis-related morbidity and mortality among patients infectedbetween the years of 1965-1985.

Fibrosis occurs as a result of a chronic toxic insult to the liver, suchas chronic hepatitis C virus (HCV) infection, autoimmune injury, andchronic exposure to toxins such as alcohol. Chronic toxic insult leadsto repeated cycles of hepatocyte injury and repair accompanied bychronic inflammation. Over a variable period of time, abnormalextracellular matrix progressively accumulates as a consequence of thehost's wound repair response. Left unchecked, this leads to increasingdeposition of fibrous material until liver architecture becomesdistorted and the liver's regenerative ability is compromised. Theprogressive accumulation of scar tissue within the liver finally resultsin the histopathologic picture of cirrhosis, defined as the formation offibrous septae throughout the liver with the formation of micronodules.

There is a need in the art for methods of treating alphavirus infectionsin general, and HCV infection in particular. The present inventionaddresses this need, and provides related advantages.

Literature

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SUMMARY OF THE INVENTION

The present invention provides methods for treating a flavivirusinfection, including hepatitis C virus (HCV) infection, in an individualsuffering from a flavivirus infection. In some embodiments, the methodsinvolve administering to an individual in need thereof an effectiveamount of an agent that inhibits enzymatic activity of a membrane-boundα-glucosidase. In other embodiments, the methods involve administeringto an individual in need thereof effective amounts of an α-glucosidaseinhibitor and at least one additional therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the effect of various amounts of Glyset® miglitolα-glucosidase inhibitor on BVDV vRNA.

FIG. 2 depicts the effect of various amounts of Precose® acarboseα-glucosidase inhibitor on BVDV vRNA.

FIG. 3 depicts the effect of various amounts of castanospermine (CTS) onBVDV vRNA.

FIG. 4 depicts the effect of various amounts of HCV NS3 inhibitorcompound BILN 2061 on BVDV vRNA.

FIG. 5 depicts the effect of various amounts of CIFN or Glyset® miglitolon BVDV vRNA.

FIG. 6 depicts the percent inhibition of BVDV treated with CIFN orGlyset® miglitol.

FIG. 7 depicts the effect of the combination of various amounts of CIFNand Glyset® miglitol on BVDV vRNA.

FIG. 8 depicts the percent inhibition of BVDV treated with a combinationof CIFN and Glyset® miglitol.

FIG. 9 is a median-effect plot of the effect of CIFN alone, Glyset®miglitol alone, or the CIFN+Glyset® miglitol combination.

FIGS. 10, 11, and 12 are conservative isobolograms for variousconcentration ranges of CIFN and Glyset® miglitol.

DEFINITIONS

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease (as in liver fibrosisthat can result in the context of chronic HCV infection); (b) inhibitingthe disease, i.e., arresting its development; and (c) relieving thedisease, i.e., causing regression of the disease.

As used herein, the term “flavivirus” includes any member of the family

Flaviviridae, including, but not limited to, Dengue virus, includingDengue virus 1, Dengue virus 2, Dengue virus 3, Dengue virus 4 (see,e.g., GenBank Accession Nos. M23027, M19197, A34774, and M14931); YellowFever Virus; West Nile Virus; Japanese Encephalitis Virus; St. LouisEncephalitis Virus; Bovine Viral Diarrhea Virus (BVDV); and Hepatitis CVirus (HCV); and any serotype, strain, genotype, subtype, quasispecies,or isolate of any of the foregoing. Where the flavivirus is HCV, the HCVis any of a number of genotypes, subtypes, or quasispecies, including,e.g., genotype 1, including 1a and 1b, 2, 3, 4, 6, etc. and subtypes(e.g., 2a, 2b, 3a, 4a, 4c, etc.), and quasispecies.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, primates, including simians and humans.

The term “treatment failure patients” (or “treatment failures”) as usedherein generally refers to HCV-infected patients who failed to respondto previous therapy for HCV (referred to as “non-responders”) or whoinitially responded to previous therapy, but in whom the therapeuticresponse was not maintained (referred to as “relapsers”). The previoustherapy generally can include treatment with IFN-α monotherapy or IFN-αcombination therapy, where the combination therapy may includeadministration of IFN-α and an antiviral agent such as ribavirin.

As used herein, the term “hepatic fibrosis,” used interchangeably hereinwith “liver fibrosis,” refers to the growth of scar tissue in the liverthat can occur in the context of a chronic hepatitis infection.

As used herein, the term “liver function” refers to a normal function ofthe liver, including, but not limited to, a synthetic function,including, but not limited to, synthesis of proteins such as serumproteins (e.g., albumin, clotting factors, alkaline phosphatase,aminotransferases (e.g., alanine transaminase, aspartate transaminase),5′-nucleosidase, γ-glutaminyltranspeptidase, etc.), synthesis ofbilirubin, synthesis of cholesterol, and synthesis of bile acids; aliver metabolic function, including, but not limited to, carbohydratemetabolism, amino acid and ammonia metabolism, hormone metabolism, andlipid metabolism; detoxification of exogenous drugs; a hemodynamicfunction, including splanchnic and portal hemodynamics; and the like.

As used herein, the term “a Type I interferon receptor agonist” refersto any naturally occurring or non-naturally occurring ligand of humanType I interferon receptor, which binds to and causes signaltransduction via the receptor. Type I interferon receptor agonistsinclude interferons, including naturally-occurring interferons, modifiedinterferons, synthetic interferons, pegylated interferons, fusionproteins comprising an interferon and a heterologous protein, shuffledinterferons; antibody specific for an interferon receptor; non-peptidechemical agonists; and the like.

As used herein, the term “HCV enzyme inhibitor” refers to any agent thatinhibits an enzymatic activity of an enzyme encoded by HCV. The term“HCV enzyme inhibitor” includes, but is not limited to, agents thatinhibit HCV NS3 protease activity; agents that inhibit HCV NS3 helicaseactivity; and agents that inhibit HCV NS5B RNA-dependent RNA polymeraseactivity.

As used herein, the terms “HCV NS3 protease inhibitor” and “NS3 proteaseinhibitor” refer to any agent that inhibits the protease activity of HCVNS3/NS4A complex. Unless otherwise specifically stated, the term “NS3inhibitor” is used interchangeably with the terms “HCV NS3 proteaseinhibitor” and “NS3 protease inhibitor.”

As used herein, the terms “HCV NS5B inhibitor,” “NS5B inhibitor,” “HCVNS5B RNA-dependent RNA polymerase inhibitor,” “HCV RDRP inhibitor,” and“RDRP inhibitor,” refer to any agent that inhibits HCV NS5BRNA-dependent RNA polymerase activity.

As used herein, the term “nucleoside” refers to a compound composed ofany pentose or modified pentose moiety attached to a specific positionof a heterocycle or to the natural position of a purine (9-position) orpyrimidine (1-position) or to the equivalent position in an analog.

As used herein, the term “nucleotide” refers to a phosphate estersubstituted on the 5′-position of a nucleoside.

As used herein, the term “heterocycle” refers to a monovalent saturatedor unsaturated carbocyclic radical having at least one hetero atom, suchas N, O, S, Se or P, within the ring, each available position of whichcan be optionally substituted, independently, with, e.g., hydroxyl, oxo,amino, irnino, lower alkyl, bromo, chloro and/or cyano. Included withinthe term “heterocycle” are purines and pyrimidines.

As used herein, the term “purine” refers to nitrogenous bicyclicheterocycles.

As used herein, the term “pyrimidine” refers to nitrogenous monocyclicheterocycles.

As used herein, the term “L-nucleoside” refers to a nucleoside compoundthat has an L-ribose sugar moiety.

As used herein, the term “a Type II interferon receptor agonist” refersto any naturally-occurring or non-naturally-occurring ligand of a humanType II interferon receptor which binds to and causes signaltransduction via the receptor. Type II interferon receptor agonistsinclude interferons, including naturally-occurring interferons, modifiedinterferons, synthetic interferons, pegylated interferons, fusionproteins comprising an interferon and a heterologous protein, shuffledinterferons; antibody specific for an interferon receptor; non-peptidechemical agonists; and the like.

As used herein, the term “alphavirus,” and its grammatical variants,refers to a group of viruses characterized by (i) an RNA genome (ii)viral replication in the cytoplasm of host cells and (iii) no DNA phaseoccurs in the viral replication cycle.

The term “hepatitis virus infection” refers to infection with one ormore of hepatitis A, B, C, D, or E virus, with blood-borne hepatitisviral infection being of particular interest, particularly hepatitis Cvirus infection.

The term “therapeutically effective amount” is meant an amount of atherapeutic agent, or a rate of delivery of a therapeutic agent,effective to facilitate a desired therapeutic effect. The precisedesired therapeutic effect will vary according to the condition to betreated, the formulation to be administered, and a variety of otherfactors that are appreciated by those of ordinary skill in the art.

The term “sustained viral response” (SVR; also referred to as a“sustained response” or a “durable response”), as used herein, refers tothe response of an individual to a treatment regimen for HCV infection,in terms of serum HCV titer. Generally, a “sustained viral response”refers to no detectable HCV RNA (e.g., less than about 500, less thanabout 200, or less than about 100 genome copies per milliliter serum)found in the patient's serum for a period of at least about one month,at least about two months, at least about three months, at least aboutfour months, at least about five months, or at least about six monthsfollowing cessation of treatment.

As used herein, the term “hepatic fibrosis,” used interchangeably hereinwith “liver fibrosis,” refers to the growth of scar tissue in the liverthat can occur in the context of a chronic hepatitis infection.

As used herein, the term “liver function” refers to a normal function ofthe liver, including, but not limited to, a synthetic function,including, but not limited to, synthesis of proteins such as serumproteins (e.g., albumin, clotting factors, alkaline phosphatase,aminotransferases (e.g., alanine transaminase, aspartate transaminase),5′-nucleosidase, γ-glutaminyltranspeptidase, etc.), synthesis ofbilirubin, synthesis of cholesterol, and synthesis of bile acids; aliver metabolic function, including, but not limited to, carbohydratemetabolism, amino acid and ammonia metabolism, hormone metabolism, andlipid metabolism; detoxification of exogenous drugs; a hemodynamicfunction, including splanchnic and portal hemodynamics; and the like.

The term “dosing event” as used herein refers to administration of anantiviral agent to a patient in need thereof, which event may encompassone or more releases of an antiviral agent from a drug dispensingdevice. Thus, the term “dosing event,” as used herein, includes, but isnot limited to, installation of a continuous delivery device (e.g., apump or other controlled release injectible system); and a singlesubcutaneous injection followed by installation of a continuous deliverysystem.

“Continuous delivery” as used herein (e.g., in the context of“continuous delivery of a substance to a tissue”) is meant to refer tomovement of drug to a delivery site, e.g., into a tissue in a fashionthat provides for delivery of a desired amount of substance into thetissue over a selected period of time, where about the same quantity ofdrug is received by the patient each minute during the selected periodof time.

“Controlled release” as used herein (e.g., in the context of “controlleddrug release”) is meant to encompass release of substance (e.g., a TypeI interferon receptor agonist, e.g., IFN-α) at a selected or otherwisecontrollable rate, interval, and/or amount, which is not substantiallyinfluenced by the environment of use. “Controlled release” thusencompasses, but is not necessarily limited to, substantially continuousdelivery, and patterned delivery (e.g., intermittent delivery over aperiod of time that is interrupted by regular or irregular timeintervals).

“Patterned” or “temporal” as used in the context of drug delivery ismeant delivery of drug in a pattern, generally a substantially regularpattern, over a pre-selected period of time (e.g., other than a periodassociated with, for example a bolus injection). “Patterned” or“temporal” drug delivery is meant to encompass delivery of drug at anincreasing, decreasing, substantially constant, or pulsatile, rate orrange of rates (e.g., amount of drug per unit time, or volume of drugformulation for a unit time), and further encompasses delivery that iscontinuous or substantially continuous, or chronic.

The term “controlled drug delivery device” is meant to encompass anydevice wherein the release (e.g., rate, timing of release) of a drug orother desired substance contained therein is controlled by or determinedby the device itself and not substantially influenced by the environmentof use, or releasing at a rate that is reproducible within theenvironment of use.

By “substantially continuous” as used in, for example, the context of“substantially continuous infusion” or “substantially continuousdelivery” is meant to refer to delivery of drug in a manner that issubstantially uninterrupted for a pre-selected period of drug delivery,where the quantity of drug, received by the patient during any 8 hourinterval in the pre-selected period never falls to zero. Furthermore,“substantially continuous” drug delivery can also encompass delivery ofdrug at a substantially constant, pre-selected rate or range of rates(e.g., amount of drug per unit time, or volume of drug formulation for aunit time) that is substantially uninterrupted for a pre-selected periodof drug delivery.

By “substantially steady state” as used in the context of a biologicalparameter that may vary as a function of time, it is meant that thebiological parameter exhibits a substantially constant value over a timecourse, such that the area under the curve defined by the value of thebiological parameter as a function of time for any 8 hour period duringthe time course (AUC8 hr) is no more than about 20% above or about 20%below, and preferably no more than about 15% above or about 15% below,and more preferably no more than about 10% above or about 10% below, theaverage area under the curve of the biological parameter over an 8 hourperiod during the time course (AUC8 hr average). The AUC8 hr average isdefined as the quotient (q) of the area under the curve of thebiological parameter over the entirety of the time course (AUCtotal)divided by the number of 8 hour intervals in the time course (ttotal1/3days), i.e., q=(AUCtotal)/ (ttotal1/3 days). For example, in the contextof a serum concentration of a drug, the serum concentration of the drugis maintained at a substantially steady state during a time course whenthe area under the curve of serum concentration of the drug over timefor any 8 hour period during the time course (AUC8 hr) is no more thanabout 20% above or about 20% below the average area under the curve ofserum concentration of the drug over an 8 hour period in the time course(AUC8 hr average), i.e., the AUC8 hr is no more than 20% above or 20%below the AUC8 hr average for the serum concentration of the drug overthe time course.

As used herein, any compound or agent described as “effective for theavoidance or amelioration of side effects induced by a Type I interferonreceptor agonist,” or as “effective for reducing or eliminating theseverity or occurrence of side effects induced by a Type I interferonreceptor agonist,” or any compound or agent described by language with ameaning similar or equivalent to that of either of the foregoing quotedpassages, is/are defined as a compound(s) or agent(s) that whenco-administered to a patient in an effective amount along with a givendosing regimen of a subject combination therapy, abates or eliminatesthe severity or occurrence of side effects experienced by a patient inresponse to the given dosing regimen of the subject combination therapy,as compared to the severity or occurrence of side effects that wouldhave been experienced by the patient in response to the same dosingregimen of the subject combination therapy without co-administration ofthe agent.

In many embodiments, the effective amounts of an inhibitor of anα-glucosidase inhibitor and a second therapeutic agent are synergisticamounts. As used herein, a “synergistic combination” or a “synergisticamount” of an inhibitor of an α-glucosidase inhibitor and a secondtherapeutic agent is a combination or amount that is more effective inthe therapeutic or prophylactic treatment of a disease than theincremental improvement in treatment outcome that could be predicted orexpected from a merely additive combination of (i) the therapeutic orprophylactic benefit of the α-glucosidase inhibitor when administered atthat same dosage as a monotherapy and (ii) the therapeutic orprophylactic benefit of the second therapeutic agent when administeredat the same dosage as a monotherapy.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anα-glucosidase inhibitor” includes a plurality of such inhibitors andreference to “the active agent” includes reference to one or more activeagents and equivalents thereof known to those skilled in the art, and soforth. It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for treating alphavirusinfections; methods of treating flavivirus infections; methods oftreating hepatitis C virus (HCV) infections; methods of treating WestNile virus (WNV) infection; methods of reducing liver fibrosis; methodsof increasing liver function in an individual suffering from liverfibrosis; methods of reducing the incidence of complications associatedwith HCV and cirrhosis of the liver; and methods of reducing viral load,or reducing the time to viral clearance, or reducing morbidity ormortality in the clinical outcomes, in patients suffering fromflavivirus infection. The methods generally involve administering to anindividual in need thereof an effective amount of an agent that inhibitsenzymatic activity of an α-glucosidase, in monotherapy or in combinationtherapy.

The methods and compositions described herein are generally useful intreatment of any alphavirus. Treatment of HCV infection is of particularinterest in some embodiments. Reference to HCV herein is forillustration only and is not meant to be limiting.

Whether a subject method is effective in treating an alphaviralinfection can be determined by a reduction in number or length ofhospital stays, a reduction in time to viral clearance, a reduction ofmorbidity or mortality in clinical outcomes, a reduction in viralburden, or other indicator of disease response in the patient.

In general, an effective amount of an agent that inhibits enzymaticactivity of an α-glucosidase is an amount that is effective to reducethe time to viral clearance, or an amount that is effective to reducemorbidity or mortality in the clinical course of the disease, or anamount that is effective to improve some other indicator of diseaseresponse (e.g., an amount that is effective to reduce viral load;achieve a sustained viral response; etc.).

In some embodiments, the present invention provides for the treatment ofan HCV infection. Whether a subject method is effective in treating anHCV infection can be determined by measuring viral load, or by measuringa parameter associated with HCV infection, including, but not limitedto, liver fibrosis, elevations in serum transaminase levels, andnecroinflammatory activity in the liver. Indicators of liver fibrosisare discussed in detail below.

Monotherapy

The present invention provides methods for treating alphavirusinfections; methods of treating flavivirus infections; methods oftreating hepatitis C virus (HCV) infections; methods of treating WestNile virus (WNV) infection; methods of reducing liver fibrosis; methodsof increasing liver function in an individual suffering from liverfibrosis; methods of reducing the incidence of complications associatedwith HCV and cirrhosis of the liver; and methods of reducing viral load,or reducing the time to viral clearance, or reducing morbidity ormortality in the clinical outcomes, in patients suffering fromflavivirus infection. The methods generally involve administering to anindividual in need thereof an effective amount of an agent that inhibitsenzymatic activity of an α-glucosidase, in monotherapy.

In these embodiments, the method involves administering an effectiveamount of an agent that inhibits enzymatic activity of a membrane-boundα-glucosidase. In some embodiments, an effective amount of an agent thatinhibits enzymatic activity of a membrane-bound α-glucosidase is anamount that is effective to reduce viral titers to undetectable levels,e.g., to about 1000 to about 5000, to about 500 to about 1000, or toabout 100 to about 500 genome copies/mL serum. In some embodiments, aneffective amount of an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase is an amount that is effective to reduceviral load to lower than 100 genome copies/mL serum.

In some embodiments, an effective amount of an agent that inhibitsenzymatic activity of a membrane-bound α-glucosidase is an amount thatis effective to achieve a 1.5-log, a 2-log, a 2.5-log, a 3-log, a3.5-log, a 4-log, a 4.5-log, or a 5-log reduction in viral titer in theserum of the individual.

Agents Suitable for use in Monotherapy

Agents that are suitable for use in a subject treatment method areagents that inhibit enzymatic activity of a membrane-bound α-glucosidehydrolase. In some embodiments, the agent is one that inhibits enzymaticactivity of a membrane-bound intestinal α-glucoside hydrolase(α-glucosidase). Membrane-bound intestinal α-glucosidases hydrolyzeoligosaccharides and disaccharides to glucose and other monosaccharidesin the brush border of the small intestine. The term “membrane-bound”refers to the association of the α-glucosidase with the plasma membraneof a cell.

Agents that are specifically excluded from use in a subject monotherapyinclude agents that inhibit an endoplasmic reticulum (ER) α-glucosidase,such as an ER α-glucosidase I, or an ER α-glucosidase II, e.g., agentsthat inhibit more than about 20% of the activity of an ER α-glucosidase.Agents that are specifically excluded from use in a subject monotherapyalso include agents that inhibit ceramide-specific glucosyltransferase(CerGlcT), e.g., agents that inhibit more than about 20% of the activityof a CerGlcT. Specific agents that are excluded from use in a subjecttreatment method include deoxynojirimycin (DNJ), deoxygalactojirimycin(DGJ), N-butyl-deoxynojirimycin (NB-DNJ), N-nonyl-deoxynojirimycin(NN-DNJ), N-butyl-deoxygalactojirimycin (NB-DGJ),N-nonyl-deoxygalactojirimycin (NN-DGJ), NN-6deoxy-DGJ, N7-oxadecyl-DNJ,N7-oxanony-6deoxy-DGJ, perbutylated-N-butyl-1-deoxynojiromycin(p-N-butyl-DNJ), and 6-O-butanoyl castanospermine.

An agent that is suitable for use in a subject monotherapy is an agentthat inhibits enzymatic activity of a membrane-bound α-glucosidase by atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, or at least about 90%, or more, compared to the enzymatic activityof the membrane-bound α-glucosidase in the absence of the agent.

A suitable agent is an agent that preferentially inhibits enzymaticactivity of a membrane-bound α-glucosidase, e.g., the agent inhibitsenzymatic activity of a membrane-bound α-glucosidase preferentially,compared to the inhibition, if any, by the agent of an ER-α-glucosidase.In other words, a suitable agent inhibits enzymatic activity of amembrane-bound α-glucosidase and if the agent inhibits anER-α-glucosidase at all, the agents inhibits less than about 20%, lessthan about 15%, less than about 10%, less than about 5%, less than about2%, or less than about 1%, of the activity of an ER-α-glucosidase.

In some embodiments, a suitable agent is a selective inhibitor of amembrane-bound α-glucosidase. The term “selective inhibitor of amembrane-bound α-glucosidase” is used herein to mean an agent whichselectively inhibits a membrane-bound α-glucosidase activity inpreference to an ER-α-glucosidase (or any other enzyme) and particularlya compound for which the ratio of the IC₅₀ concentration (concentrationinhibiting 50% of activity) for a membrane-bound α-glucosidase to theIC₅₀ concentration for an ER-α-glucosidase is greater than 1. Such ratiois readily determined by assaying for the effect of the inhibitor on amembrane-bound α-glucosidase activity and assaying for the effect of theinhibitor an ER-α-glucosidase and from the resulting data obtaining aratio of IC₅₀s.

Of particular interest in some embodiments of a subject monotherapy isuse of an agent that inhibits enzymatic activity of a membrane-boundα-glucosidase with an IC50 of less than about 50 μM, e.g., a suitableagent inhibits enzymatic activity of a membrane-bound α-glucosidase withan IC50 of less than about 40 μM, less than about 25 μM, less than about10 μM, less than about 1 μM, less than about 100 nM, less than about 80nM, less than about 60 nM, less than about 50 nM, less than about 25 nM,less than about 10 nM, or less than about 1 nM, or less.

In many embodiments, an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase inhibits viral replication. For example, anagent that inhibits enzymatic activity of a membrane-bound α-glucosidaseinhibits viral replication by at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, or at least about 90%, or more, compared to viralreplication in the absence of the compound. Whether a compound inhibitsviral replication can be determined using methods known in the art,including an in vitro viral replication assay.

In some embodiments, the agent is an imino sugar. In some embodiments,the agent is miglitol (3,4,5-piperidinetriol,1-(2-hydroxyethyl)-2-(hydroxymethyl)-, [2R-(2α, 3β, 4α, 5β)]) or Glyset®(miglitol; N-hydroxyethyl-DNJ). Miglitol (N-hydroxyethyl-DNJ) isdescribed in U.S. Pat. No. 4,639,436. Miglitol has the structure shownin Formula I:

In some embodiments, the agent is acarbose(O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-1-yl]amino]-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucose),or Precose®. Acarbose is described in U.S. Pat. No. 4,904,769. In someembodiments, acarbose is a highly purified form of acarbose (see, e.g.,U.S. Pat. No. 4,904,769). Acarbose has the structure shown in FormulaII:

Hepatitis Virus Infection

The present invention provides monotherapy methods of treating ahepatitis virus infection. In particular embodiments, the presentinvention provides methods of treating a hepatitis C virus (HCV)infection; methods of reducing the incidence of complications associatedwith HCV and cirrhosis of the liver; and methods of reducing viral load,or reducing the time to viral clearance, or reducing morbidity ormortality in the clinical outcomes, in patients suffering from HCVinfection. The methods generally involve administering to the individualan effective amount of an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase in monotherapy. Effective amounts of anagent that inhibits enzymatic activity of a membrane-boundα-glucosidase, as well as dosing regimens, are discussed below.

In many embodiments, a subject monotherapy treatment method is effectiveto decrease viral load in the individual, and to achieve a sustainedviral response. Of particular interest in many embodiments is treatmentof humans.

Whether a subject monotherapy method is effective in treating an HCVinfection can be determined by measuring viral load, or by measuring aparameter associated with HCV infection, including, but not limited to,liver fibrosis, elevations in serum transaminase levels, andnecroinflammatory activity in the liver. Indicators of liver fibrosisare discussed in detail below.

Viral load can be measured by measuring the titer or level of virus inserum.

These methods include, but are not limited to, a quantitative polymerasechain reaction (PCR) and a branched DNA (bDNA) test. Quantitative assaysfor measuring the viral load (titer) of HCV RNA have been developed.Many such assays are available commercially, including a quantitativereverse transcription PCR (RT-PCR) (Amplicor HCV Monitor™, RocheMolecular Systems, New Jersey); and a branched DNA (deoxyribonucleicacid) signal amplification assay (Quantiplex™ HCV RNA Assay (bDNA),Chiron Corp., Emeryville, Calif.). See, e.g., Gretch et al. (1995) Ann.Intern. Med. 123:321-329. Also of interest is a nucleic acid test (NAT),developed by Gen-Probe Inc. (San Diego) and Chiron Corporation, and soldby Chiron Corporation under the trade name Procleix®, which NATsimultaneously tests for the presence of HIV-1 and HCV. See, e.g., Vargoet al. (2002) Transfusion 42:876-885.

In some embodiments, an effective amount of an agent that inhibitsenzymatic activity of a membrane-bound α-glucosidase is an amount that,in monotherapy, is effective to reduce HCV viral load to undetectablelevels, e.g., to less than about 5000, less than about 1000, less thanabout 500, or less than about 200 genome copies/mL serum. In someembodiments, an effective amount of an agent that inhibits enzymaticactivity of a membrane-bound α-glucosidase is an amount that, inmonotherapy, is effective to reduce HCV viral load to less than 100genome copies/mL serum.

In other embodiments, an effective amount of an agent that inhibitsenzymatic activity of a membrane-bound α-glucosidase is an amount that,in monotherapy, is effective to achieve a 1.5-log, a 2-log, a 2.5-log, a3-log, a 3.5-log, a 4-log, a 4.5-log, or a 5-log reduction in HCV viraltiter in the serum of the individual.

In many embodiments, the methods of the invention achieve a sustainedviral response, e.g., the viral load is reduced to undetectable levelsfor a period of at least about one month, at least about two months, atleast about three months, at least about four months, at least aboutfive months, or at least about six months following cessation oftreatment.

Whether a subject method is effective in treating an HCV infection canbe determined by measuring a parameter associated with HCV infection,such as liver fibrosis. Methods of determining the extent of liverfibrosis are discussed in detail below. In some embodiments, the levelof a serum marker of liver fibrosis indicates the degree of liverfibrosis.

As one non-limiting example, levels of serum alanine aminotransferase(ALT) are measured, using standard assays. In general, an ALT level ofless than about 45 international units is considered normal. In someembodiments, an effective amount of a an agent that inhibits enzymaticactivity of a membrane-bound α-glucosidase that is administered in asubject monotherapy is an amount effective to reduce ALT levels to lessthan about 45 U/ml serum.

In other embodiments, a therapeutically effective amount of an agentthat inhibits enzymatic activity of a membrane-bound α-glucosidase is anamount that, in monotherapy, is effective to reduce a serum level of amarker of liver fibrosis by at least about 10%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, or at least about 80%, or more, compared to the level of the markerin an untreated individual, or to a placebo-treated individual. Methodsof measuring serum markers include immunological-based methods, e.g.,enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, and thelike, using antibody specific for a given serum marker.

West Nile Virus

The present invention provides monotherapy methods for treating WestNile viral infection. The methods generally involve administering to anindividual an agent that inhibits enzymatic activity of a membrane-boundα-glucosidase in an amount that is effective to reduce the time to viralclearance in the individual, and/or to ameliorate the clinical course ofthe disease. Effective amounts of an agent that inhibits enzymaticactivity of a membrane-bound α-glucosidase, as well as dosing regimens,are discussed below.

Whether a subject method is effective in treating a West Nile viralinfection can be determined by a reduction in number or length ofhospital stays, a reduction in time to viral clearance, a reduction ofmorbidity or mortality in clinical outcomes, or other indicator ofdisease response.

In some embodiments, an effective amount of an agent that inhibitsenzymatic activity of a membrane-bound α-glucosidase is an amount that,in monotherapy, is effective to reduce the time to viral clearance, oran amount that is effective to reduce morbidity or mortality in theclinical course of the disease.

Liver Fibrosis

The instant invention provides monotherapy methods for treating liverfibrosis (including forms of liver fibrosis resulting from, orassociated with, HCV infection), generally involving administering atherapeutic amount of an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase. Effective amounts of an agent thatinhibits enzymatic activity of a membrane-bound α-glucosidase, as wellas dosing regimens, are discussed below.

Whether treatment with an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase is effective in reducing liver fibrosis canbe determined by any of a number of well-established techniques formeasuring liver fibrosis and liver function. Liver fibrosis reduction isdetermined by analyzing a liver biopsy sample. An analysis of a liverbiopsy comprises assessments of two major components: necroinflammationassessed by “grade” as a measure of the severity and ongoing diseaseactivity, and the lesions of fibrosis and parenchymal or vascularremodeling as assessed by “stage” as being reflective of long-termdisease progression. See, e.g., Brunt (2000) Hepatol. 31:241-246; andMETAVIR (1994) Hepatology 20:15-20. Based on analysis of the liverbiopsy, a score is assigned. A number of standardized scoring systemsexist which provide a quantitative assessment of the degree and severityof fibrosis. These include the METAVIR, Knodell, Scheuer, Ludwig, andIshak scoring systems.

The METAVIR scoring system is based on an analysis of various featuresof a liver biopsy, including fibrosis (portal fibrosis, centrilobularfibrosis, and cirrhosis); necrosis (piecemeal and lobular necrosis,acidophilic retraction, and ballooning degeneration); inflammation(portal tract inflammation, portal lymphoid aggregates, and distributionof portal inflammation); bile duct changes; and the Knodell index(scores of periportal necrosis, lobular necrosis, portal inflammation,fibrosis, and overall disease activity). The definitions of each stagein the METAVIR system are as follows: score: 0, no fibrosis; score: 1,stellate enlargement of portal tract but without septa formation; score:2, enlargement of portal tract with rare septa formation; score: 3,numerous septa without cirrhosis; and score: 4, cirrhosis.

Knodell's scoring system, also called the Hepatitis Activity Index,classifies specimens based on scores in four categories of histologicfeatures: I. Periportal and/or bridging necrosis; II. Intralobulardegeneration and focal necrosis; III. Portal inflammation; and IV.Fibrosis. In the Knodell staging system, scores are as follows: score:0, no fibrosis; score: 1, mild fibrosis (fibrous portal expansion);score: 2, moderate fibrosis; score: 3, severe fibrosis (bridgingfibrosis); and score: 4, cirrhosis. The higher the score, the moresevere the liver tissue damage. Knodell (1981) Hepatol. 1:431.

In the Scheuer scoring system scores are as follows: score: 0, nofibrosis; score:

1, enlarged, fibrotic portal tracts; score: 2, periportal orportal-portal septa, but intact architecture; score: 3, fibrosis witharchitectural distortion, but no obvious cirrhosis; score: 4, probableor definite cirrhosis. Scheuer (1991) J. Hepatol. 13:372.

The Ishak scoring system is described in Ishak (1995) J. Hepatol.22:696-699. Stage 0, No fibrosis; Stage 1, Fibrous expansion of someportal areas, with or without short fibrous septa; stage 2, Fibrousexpansion of most portal areas, with or without short fibrous septa;stage 3, Fibrous expansion of most portal areas with occasional portalto portal (P-P) bridging; stage 4, Fibrous expansion of portal areaswith marked bridging (P-P) as well as portal-central (P-C); stage 5,Marked bridging (P-P and/or P-C) with occasional nodules (incompletecirrhosis); stage 6, Cirrhosis, probable or definite.

The benefit of a subject therapy can also be measured and assessed byusing the

Child-Pugh scoring system which comprises a multicomponent point systembased upon abnormalities in serum bilirubin level, serum albumin level,prothrombin time, the presence and severity of ascites, and the presenceand severity of encephalopathy. Based upon the presence and severity ofabnormality of these parameters, patients may be placed in one of threecategories of increasing severity of clinical disease: A, B, or C.

In some embodiments, an effective amount of an agent that inhibitsenzymatic activity of a membrane-bound α-glucosidase is an amount of anagent that inhibits enzymatic activity of a membrane-bound α-glucosidaseand that effects a change of one unit or more in the fibrosis stagebased on pre- and post-therapy liver biopsies. In particularembodiments, a therapeutically effective amount of an agent thatinhibits enzymatic activity of a membrane-bound α-glucosidase reducesliver fibrosis by at least one unit in the METAVIR, the Knodell, theScheuer, the Ludwig, or the Ishak scoring system.

Secondary, or indirect, indices of liver function can also be used toevaluate the efficacy of a subject treatment. Morphometric computerizedsemi-automated assessment of the quantitative degree of liver fibrosisbased upon specific staining of collagen and/or serum markers of liverfibrosis can also be measured as an indication of the efficacy of asubject treatment method. Secondary indices of liver function include,but are not limited to, serum transaminase levels, prothrombin time,bilirubin, platelet count, portal pressure, albumin level, andassessment of the Child-Pugh score.

In other embodiments, an effective amount of an agent that inhibitsenzymatic activity of a membrane-bound u-glucosidase is an amount thatis effective to increase an index of liver function by at least about10%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, or at least about 80%, or more,compared to the index of liver function in an untreated individual, orto a placebo-treated individual. Those skilled in the art can readilymeasure such indices of liver function, using standard assay methods,many of which are commercially available, and are used routinely inclinical settings.

Serum markers of liver fibrosis can also be measured as an indication ofthe efficacy of a subject treatment method. Serum markers of liverfibrosis include, but are not limited to, hyaluronate, N-terminalprocollagen III peptide, 7S domain of type IV collagen, C-terminalprocollagen I peptide, and laminin. Additional biochemical markers ofliver fibrosis include α-2-macroglobulin, haptoglobin, gamma globulin,apolipoprotein A, and gamma glutamyl transpeptidase.

In other embodiments, a therapeutically effective amount of an agentthat inhibits enzymatic activity of a membrane-bound α-glucosidase is anamount that, in monotherapy, is effective to reduce a serum level of amarker of liver fibrosis by at least about 10%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, or at least about 80%, or more, compared to the level of the markerin an untreated individual, or to a placebo-treated individual. Thoseskilled in the art can readily measure such serum markers of liverfibrosis, using standard assay methods, many of which are commerciallyavailable, and are used routinely in clinical settings. Methods ofmeasuring serum markers include immunological-based methods, e.g.,enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, and thelike, using antibody specific for a given serum marker.

Quantitative tests of functional liver reserve can also be used toassess the efficacy of treatment with an agent that inhibits enzymaticacitivty of a membrane-bound α-glucosidase. These include: indocyaninegreen clearance (ICG), galactose elimination capacity (GEC), aminopyrinebreath test (ABT), antipyrine clearance, monoethylglycine-xylidide(MEG-X) clearance, and caffeine clearance.

As used herein, a “complication associated with cirrhosis of the liver”refers to a disorder that is a sequellae of decompensated liver disease,i.e., or occurs subsequently to and as a result of development of liverfibrosis, and includes, but it not limited to, development of ascites,variceal bleeding, portal hypertension, jaundice, progressive liverinsufficiency, encephalopathy, hepatocellular carcinoma, liver failurerequiring liver transplantation, and liver-related mortality.

In other embodiments, a therapeutically effective amount of an agentthat inhibits enzymatic activity of a membrane-bound α-glucosidase is anamount that is effective in reducing the incidence (e.g., the likelihoodthat an individual will develop) of a disorder associated with cirrhosisof the liver by at least about 10%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, or atleast about 80%, or more, compared to an untreated individual, or to aplacebo-treated individual.

Whether treatment with an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase is effective in reducing the incidence of adisorder associated with cirrhosis of the liver can readily bedetermined by those skilled in the art.

Reduction in liver fibrosis increases liver function. Thus, theinvention provides methods for increasing liver function, generallyinvolving administering a therapeutically effective amount of an agentthat inhibits enzymatic activity of a membrane-bound α-glucosidase.Liver functions include, but are not limited to, synthesis of proteinssuch as serum proteins (e.g., albumin, clotting factors, alkalinephosphatase, aminotransferases (e.g., alanine transaminase, aspartatetransaminase), 5′-nucleosidase, γ-glutaminyltranspeptidase, etc.),synthesis of bilirubin, synthesis of cholesterol, and synthesis of bileacids; a liver metabolic function, including, but not limited to,carbohydrate metabolism, amino acid and ammonia metabolism, hormonemetabolism, and lipid metabolism; detoxification of exogenous drugs; ahemodynamic function, including splanchnic and portal hemodynamics; andthe like.

Whether a liver function is increased is readily ascertainable by thoseskilled in the art, using well-established tests of liver function.Thus, synthesis of markers of liver function such as albumin, alkalinephosphatase, alanine transaminase, aspartate transaminase, bilirubin,and the like, can be assessed by measuring the level of these markers inthe serum, using standard immunological and enzymatic assays. Splanchniccirculation and portal hemodynamics can be measured by portal wedgepressure and/or resistance using standard methods. Metabolic functionscan be measured by measuring the level of ammonia in the serum.

Whether serum proteins normally secreted by the liver are in the normalrange can be determined by measuring the levels of such proteins, usingstandard immunological and enzymatic assays. Those skilled in the artknow the normal ranges for such serum proteins. The following arenon-limiting examples. The normal level of alanine transaminase is about45 IU per milliliter of serum. The normal range of aspartatetransaminase is from about 5 to about 40 units per liter of serum.Bilirubin is measured using standard assays. Normal bilirubin levels areusually less than about 1.2 mg/dL. Serum albumin levels are measuredusing standard assays. Normal levels of serum albumin are in the rangeof from about 35 to about 55 g/L. Prolongation of prothrombin time ismeasured using standard assays. Normal prothrombin time is less thanabout 4 seconds longer than control.

In some embodiments, a therapeutically effective amount of an agent thatinhibits enzymatic activity of a membrane-bound α-glucosidase is onethat is effective to increase liver function by at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, ormore. In other embodiments, a therapeutically effective amount of anagent that inhibits enzymatic activity of a membrane-bound α-glucosidaseis an amount effective to reduce an elevated level of a serum marker ofliver function by at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, or more, or to reduce the level ofthe serum marker of liver function to within a normal range. In otherembodiments, a therapeutically effective amount of an agent thatinhibits enzymatic activity of a membrane-bound α-glucosidase is anamount effective to increase a reduced level of a serum marker of liverfunction by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or more, or to increase the level of theserum marker of liver function to within a normal range.

Combination Therapy

The present invention further provides combination therapies. Thus, thepresent invention provides methods for treating alphavirus infections;methods of treating flavivirus infections; methods of treating HCVinfections; methods of treating WNV infection; methods of reducing liverfibrosis; methods of increasing liver function in an individualsuffering from liver fibrosis; methods of reducing the incidence ofcomplications associated with HCV and cirrhosis of the liver; andmethods of reducing viral load, or reducing the time to viral clearance,or reducing morbidity or mortality in the clinical outcomes, in patientssuffering from flavivirus infection. The methods generally involveadministering to an individual in need thereof an effective amount of anagent that inhibits enzymatic activity of an α-glucosidase, incombination with at least a second therapeutic agent. In someembodiments, the effective amounts of the α-glucosidase inhibitor andthe at least one additional therapeutic agent are synergistic amounts.

Suitable second therapeutic agents for treating an alphavirus infectioninclude, but are not limited to, a Type I interferon receptor agonist, aType II interferon receptor agonist; a Type III interferon receptoragonist; a nucleoside analog (e.g., ribavirin, levovirin, orviramidine); an NS3 protease inhibitor, and NS3 helicase inhibitor, anNS5B inhibitor, and thymosin-α.

A subject combination therapy involves administering effective amountsof an α-glucosidase inhibitor and at least a second therapeutic agent.In some embodiments, effective amounts of an α-glucosidase inhibitor andat least a second therapeutic agent are amounts that, in combinationtherapy, are effective to reduce viral titers to undetectable levels,e.g., to about 1000 to about 5000, to about 500 to about 1000, or toabout 100 to about 500 genome copies/mL serum. In some embodiments,effective amounts of an α-glucosidase inhibitor and at least a secondtherapeutic agent are amounts that, in combination therapy, areeffective to reduce viral load to lower than 100 genome copies/mL serum.

In some embodiments, effective amounts of an α-glucosidase inhibitor andat least a second therapeutic agent are amounts that, in combinationtherapy, are effective to achieve a 1.5-log, a 2-log, a 2.5-log, a3-log, a 3.5-log, a 4-log, a 4.5-log, or a 5-log reduction in viraltiter in the serum of the individual.

In some embodiments, effective amounts of an α-glucosidase inhibitor andat least a second therapeutic agent are amounts that, in combinationtherapy, are synergistic amounts. In some of these embodiments, theamount of the at least one second therapeutic agent that is required tobe administered to the individual to achieve a desired therapeuticeffect (e.g., reduction in serum viral load) is reduced, compared to thedose that is normally required to be administered to achieve the sameeffect (e.g., the same reduction in serum viral load), when the at leastone additional second therapeutic agent is administered in monotherapyor in the absence of co-administration with an α-glucosidase inhibitor.As one non-limiting example, a reduction in HCV serum viral load can beachieved using a combination of α-glucosidase inhibitor and consensusIFN-α (CIFN), where the amount of CIFN in the combination therapy thatis required to achieve the reduction in the serum HCV viral load islower than the amount of CIFN that would be required to achieve the samereduction in HCV serum viral load were the CIFN administered inmonotherapy. In some of these α-glucosidase inhibitor/second therapeuticagent combination therapy embodiments, the amount of the α-glucosidaseinhibitor that is required to be administered to the individual toachieve a desired therapeutic effect (e.g., reduction in serum viralload) is reduced, compared to the dose that is normally required to beadministered to achieve the same effect (e.g., the same reduction inserum viral load), when the α-glucosidase inhibitor is administered inmonotherapy. In some of these α-glucosidase inhibitor/second therapeuticagent combination therapy embodiments, the amount of the α-glucosidaseinhibitor and the amount of the second therapeutic agent that arerequired to be administered to the individual to achieve a desiredtherapeutic effect (e.g., reduction in serum viral load) are reduced,compared to the doses that are required to achieve the same effect(e.g., the same reduction in serum viral load) in monotherapy, e.g.,when the α-glucosidase inhibitor is administered in monotherapy, andwhen the second therapeutic agent is administered in monotherapy.

Hepatitis Virus Infection

The present invention provides combination therapy methods of treating ahepatitis virus infection. In particular embodiments, the presentinvention provides methods of treating a hepatitis C virus (HCV)infection; methods of reducing the incidence of complications associatedwith HCV and cirrhosis of the liver; and methods of reducing viral load,or reducing the time to viral clearance, or reducing morbidity ormortality in the clinical outcomes, in patients suffering from HCVinfection. The methods generally involve administering to the individualcombined effective amounts of an α-glucosidase inhibitor and at least asecond therapeutic agent. Combined effective amounts of an α-glucosidaseinhibitor and at least a second therapeutic agent, as well as dosingregimens, are discussed below.

In many embodiments, a subject combination treatment method is effectiveto decrease viral load in the individual, and to achieve a sustainedviral response. Of particular interest in many embodiments is treatmentof humans.

Whether a subject combination method is effective in treating an HCVinfection can be determined by measuring viral load, or by measuring aparameter associated with HCV infection, including, but not limited to,liver fibrosis, elevations in serum transaminase levels, andnecroinflammatory activity in the liver. Indicators of liver fibrosisare discussed in detail below.

Viral load can be measured by measuring the titer or level of virus inserum. These methods include, but are not limited to, a quantitativepolymerase chain reaction (PCR) and a branched DNA (bDNA) test.Quantitative assays for measuring the viral load (titer) of HCV RNA havebeen developed. Many such assays are available commercially, including aquantitative reverse transcription PCR (RT-PCR) (Amplicor HCV Monitor™,Roche Molecular Systems, New Jersey); and a branched DNA(deoxyribonucleic acid) signal amplification assay (Quantiplex™ HCV RNAAssay (bDNA), Chiron Corp., Emeryville, Calif.). See, e.g., Gretch etal. (1995) Ann. Intern. Med. 123:321-329. Also of interest is a nucleicacid test (NAT), developed by Gen-Probe Inc. (San Diego) and ChironCorporation, and sold by Chiron Corporation under the trade nameProcleix®, which NAT simultaneously tests for the presence of HIV-1 andHCV. See, e.g., Vargo et al. (2002) Transfusion 42:876-885.

In some embodiments, effective amounts of an α-glucosidase inhibitor andat least a second therapeutic agent are amounts that, in combinationtherapy, are effective to reduce HCV viral load to undetectable levels,e.g., to less than about 5000, less than about 1000, less than about500, or less than about 200 genome copies/mL serum. In some embodiments,effective amounts of an α-glucosidase inhibitor and at least a secondtherapeutic agent are amounts that, in combination therapy, areeffective to reduce HCV viral load to less than 100 genome copies/mLserum.

In other embodiments, effective amounts of an α-glucosidase inhibitorand at least a second therapeutic agent are amounts that, in combinationtherapy, are effective to achieve a 1.5-log, a 2-log, a 2.5-log, a3-log, a 3.5-log, a 4-log, a 4.5-log, or a 5-log reduction in HCV viraltiter in the serum of the individual.

In many embodiments, the methods of the invention achieve a sustainedviral response, e.g., the viral load is reduced to undetectable levelsfor a period of at least about one month, at least about two months, atleast about three months, at least about four months, at least aboutfive months, or at least about six months following cessation oftreatment.

Whether a subject method is effective in treating an HCV infection canbe determined by measuring a parameter associated with HCV infection,such as liver fibrosis. Methods of determining the extent of liverfibrosis are discussed in detail below. In some embodiments, the levelof a serum marker of liver fibrosis indicates the degree of liverfibrosis.

As one non-limiting example, levels of serum alanine aminotransferase(ALT) are measured, using standard assays. In general, an ALT level ofless than about 45 international units is considered normal. In someembodiments, an effective amount of a therapeutic agent that isadministered as part of a subject combination therapy is an amounteffective to reduce ALT levels to less than about 45 U/ml serum.

In other emobidments, effective amounts of an α-glucosidase inhibitorand at least a second therapeutic agent are amounts that, in combinationtherapy, are effective to reduce a serum level of a marker of liverfibrosis by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, or at leastabout 80%, or more, compared to the level of the marker in an untreatedindividual, or to a placebo-treated individual. Methods of measuringserum markers include immunological-based methods, e.g., enzyme-linkedimmunosorbent assays (ELISA), radioimmunoassays, and the like, usingantibody specific for a given serum marker.

West Nile Virus

The present invention provides combination therapy methods for treatingWest Nile viral infection. The methods generally involve administeringto an individual an α-glucosidase inhibitor and at least a secondtherapeutic agent in amounts that in amounts that in combination therapyare effective to reduce the time to viral clearance in the individual,and/or to ameliorate the clinical course of the disease. Effectiveamounts of an α-glucosidase inhibitor and at least a second therapeuticagent, as well as dosing regimens, are discussed below.

Whether a subject method is effective in treating a West Nile viralinfections can be determined by a reduction in number or length ofhospital stays, a reduction in time to viral clearance, a reduction ofmorbidity or mortality in clinical outcomes, or other indicator ofdisease response.

In some embodiments, effective amounts of an α-glucosidase inhibitor andat least a second therapeutic agent are amounts that, in combinationtherapy, are effective to reduce the time to viral clearance, or anamount that is effective to reduce morbidity or mortality in theclinical course of the disease.

Liver Fibrosis

The instant invention provides combination therapy methods for treatingliver fibrosis (including forms of liver fibrosis resulting from, orassociated with, HCV infection), generally involving administeringeffective amounts of an α-glucosidase inhibitor and at least a secondtherapeutic agent. Effective amounts of an α-glucosidase inhibitor andat least a second therapeutic agent, as well as dosing regimens, arediscussed below.

Whether a subject combination therapy is effective in reducing liverfibrosis can be determined by any of a number of well-establishedtechniques for measuring liver fibrosis and liver function. Liverfibrosis reduction is determined by analyzing a liver biopsy sample. Ananalysis of a liver biopsy comprises assessments of two majorcomponents: necroinflammation assessed by “grade” as a measure of theseverity and ongoing disease activity, and the lesions of fibrosis andparenchymal or vascular remodeling as assessed by “stage” as beingreflective of long-term disease progression. See, e.g., Brunt (2000)Hepatol. 31:241-246; and METAVIR (1994) Hepatology 20:15-20. Based onanalysis of the liver biopsy, a score is assigned. A number ofstandardized scoring systems exist which provide a quantitativeassessment of the degree and severity of fibrosis. These include theMETAVIR, Knodell, Scheuer, Ludwig, and Ishak scoring systems.

The METAVIR scoring system is based on an analysis of various featuresof a liver biopsy, including fibrosis (portal fibrosis, centrilobularfibrosis, and cirrhosis); necrosis (piecemeal and lobular necrosis,acidophilic retraction, and ballooning degeneration); inflammation(portal tract inflammation, portal lymphoid aggregates, and distributionof portal inflammation); bile duct changes; and the Knodell index(scores of periportal necrosis, lobular necrosis, portal inflammation,fibrosis, and overall disease activity). The definitions of each stagein the METAVIR system are as follows: score: 0, no fibrosis; score: 1,stellate enlargement of portal tract but without septa formation; score:2, enlargement of portal tract with rare septa formation; score: 3,numerous septa without cirrhosis; and score: 4, cirrhosis.

Knodell's scoring system, also called the Hepatitis Activity Index,classifies specimens based on scores in four categories of histologicfeatures: I. Periportal and/or bridging necrosis; II. Intralobulardegeneration and focal necrosis; III. Portal inflammation; and IV.Fibrosis. In the Knodell staging system, scores are as follows: score:0, no fibrosis; score: 1, mild fibrosis (fibrous portal expansion);score: 2, moderate fibrosis; score: 3, severe fibrosis (bridgingfibrosis); and score: 4, cirrhosis. The higher the score, the moresevere the liver tissue damage. Knodell (1981) Hepatol. 1:431.

In the Scheuer scoring system scores are as follows: score: 0, nofibrosis; score: 1, enlarged, fibrotic portal tracts; score: 2,periportal or portal-portal septa, but intact architecture; score: 3,fibrosis with architectural distortion, but no obvious cirrhosis; score:4, probable or definite cirrhosis. Scheuer (1991) J. Hepatol. 13:372.

The Ishak scoring system is described in Ishak (1995) J. Hepatol.22:696-699. Stage 0, No fibrosis; Stage 1, Fibrous expansion of someportal areas, with or without short fibrous septa; stage 2, Fibrousexpansion of most portal areas, with or without short fibrous septa;stage 3, Fibrous expansion of most portal areas with occasional portalto portal (P-P) bridging; stage 4, Fibrous expansion of portal areaswith marked bridging (P-P) as well as portal-central (P-C); stage 5,Marked bridging (P-P and/or P-C) with occasional nodules (incompletecirrhosis); stage 6, Cirrhosis, probable or definite.

The benefit of a subject combination therapy can also be measured andassessed by using the Child-Pugh scoring system which comprises amulticomponent point system based upon abnormalities in serum bilirubinlevel, serum albumin level, prothrombin time, the presence and severityof ascites, and the presence and severity of encephalopathy. Based uponthe presence and severity of abnormality of these parameters, patientsmay be placed in one of three categories of increasing severity ofclinical disease: A, B, or C.

In some embodiments, effective amounts of an α-glucosidase inhibitor andat least a second therapeutic agent are amounts that, in combinationtherapy, are amounts that effect a change of one unit or more in thefibrosis stage based on pre- and post-therapy liver biopsies. Inparticular embodiments, therapeutically effective amounts of anα-glucosidase inhibitor and at least a second therapeutic agent areamounts that, in combination therapy, reduce liver fibrosis by at leastone unit in the METAVIR, the Knodell, the Scheuer, the Ludwig, or theIshak scoring system.

Secondary, or indirect, indices of liver function can also be used toevaluate the efficacy of a subject treatment. Morphometric computerizedsemi-automated assessment of the quantitative degree of liver fibrosisbased upon specific staining of collagen and/or serum markers of liverfibrosis can also be measured as an indication of the efficacy of asubject treatment method. Secondary indices of liver function include,but are not limited to, serum transaminase levels, prothrombin time,bilirubin, platelet count, portal pressure, albumin level, andassessment of the Child-Pugh score.

In other embodiments, effective amounts of an α-glucosidase inhibitorand at least a second therapeutic agent are amounts that, in combinationtherapy, are effective to increase an index of liver function by atleast about 10%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, or at least about 80%, ormore, compared to the index of liver function in an untreatedindividual, or to a placebo-treated individual. Those skilled in the artcan readily measure such indices of liver function, using standard assaymethods, many of which are commercially available, and are usedroutinely in clinical settings.

Serum markers of liver fibrosis can also be measured as an indication ofthe efficacy of a subject treatment method. Serum markers of liverfibrosis include, but are not limited to, hyaluronate, N-terminalprocollagen III peptide, 7S domain of type IV collagen, C-terminalprocollagen I peptide, and laminin. Additional biochemical markers ofliver fibrosis include α-2-macroglobulin, haptoglobin, gamma globulin,apolipoprotein A, and gamma glutamyl transpeptidase.

In other embodiments, effective amounts of an α-glucosidase inhibitorand at least a second therapeutic agent are amounts that, in combinationtherapy, are effective to reduce a serum level of a marker of liverfibrosis by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, or at leastabout 80%, or more, compared to the level of the marker in an untreatedindividual, or to a placebo-treated individual. Those skilled in the artcan readily measure such serum markers of liver fibrosis, using standardassay methods, many of which are commercially available, and are usedroutinely in clinical settings. Methods of measuring serum markersinclude immunological-based methods, e.g., enzyme-linked immunosorbentassays (ELISA), radioimmunoassays, and the like, using antibody specificfor a given serum marker.

Quantitative tests of functional liver reserve can also be used toassess the efficacy of treatment with an agent that inhibits enzymaticacitivty of a membrane-bound α-glucosidase. These include: indocyaninegreen clearance (ICG), galactose elimination capacity (GEC), aminopyrinebreath test (ABT), antipyrine clearance, monoethylglycine-xylidide(MEG-X) clearance, and caffeine clearance.

As used herein, a “complication associated with cirrhosis of the liver”refers to a disorder that is a sequellae of decompensated liver disease,i.e., or occurs subsequently to and as a result of development of liverfibrosis, and includes, but it not limited to, development of ascites,variceal bleeding, portal hypertension, jaundice, progressive liverinsufficiency, encephalopathy, hepatocellular carcinoma, liver failurerequiring liver transplantation, and liver-related mortality.

In other embodiments, effective amounts of an α-glucosidase inhibitorand at least a second therapeutic agent are amounts that, in combinationtherapy, are effective in reducing the incidence (e.g., the likelihoodthat an individual will develop) of a disorder associated with cirrhosisof the liver by at least about 10%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, or atleast about 80%, or more, compared to an untreated individual, or to aplacebo-treated individual.

Whether treatment with an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase is effective in reducing the incidence of adisorder associated with cirrhosis of the liver can readily bedetermined by those skilled in the art.

Reduction in liver fibrosis increases liver function. Thus, theinvention provides methods for increasing liver function, generallyinvolving administering a therapeutically effective amount of an agentthat inhibits enzymatic activity of a membrane-bound α-glucosidase.Liver functions include, but are not limited to, synthesis of proteinssuch as serum proteins (e.g., albumin, clotting factors, alkalinephosphatase, aminotransferases (e.g., alanine transaminase, aspartatetransaminase), 5′-nucleosidase, γ-glutaminyltranspeptidase, etc.),synthesis of bilirubin, synthesis of cholesterol, and synthesis of bileacids; a liver metabolic function, including, but not limited to,carbohydrate metabolism, amino acid and ammonia metabolism, hormonemetabolism, and lipid metabolism; detoxification of exogenous drugs; ahemodynamic function, including splanchnic and portal hemodynamics; andthe like.

Whether a liver function is increased is readily ascertainable by thoseskilled in the art, using well-established tests of liver function.Thus, synthesis of markers of liver function such as albumin, alkalinephosphatase, alanine transaminase, aspartate transaminase, bilirubin,and the like, can be assessed by measuring the level of these markers inthe serum, using standard immunological and enzymatic assays. Splanchniccirculation and portal hemodynamics can be measured by portal wedgepressure and/or resistance using standard methods. Metabolic functionscan be measured by measuring the level of ammonia in the serum.

Whether serum proteins normally secreted by the liver are in the normalrange can be determined by measuring the levels of such proteins, usingstandard immunological and enzymatic assays. Those skilled in the artknow the normal ranges for such serum proteins. The following arenon-limiting examples. The normal level of alanine transaminase is about45 IU per milliliter of serum. The normal range of aspartatetransaminase is from about 5 to about 40 units per liter of serum.Bilirubin is measured using standard assays. Normal bilirubin levels areusually less than about 1.2 mg/dL. Serum albumin levels are measuredusing standard assays. Normal levels of serum albumin are in the rangeof from about 35 to about 55 g/L. Prolongation of prothrombin time ismeasured using standard assays. Normal prothrombin time is less thanabout 4 seconds longer than control.

In some embodiments, effective amounts of an α-glucosidase inhibitor andat least a second therapeutic agent are amounts that, in combinationtherapy, are effective to increase liver function by at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,or more. In other embodiments, effective amounts of an α-glucosidaseinhibitor and at least a second therapeutic agent are amounts that, incombination therapy, are effective to reduce an elevated level of aserum marker of liver function by at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, or more, or toreduce the level of the serum marker of liver function to within anormal range. In other embodiments, effective amounts of anα-glucosidase inhibitor and at least a second therapeutic agent areamounts that, in combination therapy, are effective to increase areduced level of a serum marker of liver function by at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,or more, or to increase the level of the serum marker of liver functionto within a normal range.

Alphα-glucosidase Inhibitors

Alphα-glucosidase inhibitors suitable for use in a subject combinationtherapy include any imino-sugar, including long-alkyl chain derivativesof imino sugars as disclosed in U.S. Patent Publication No.2004/0110795; inhibitors of endoplasmic reticulum-associatedα-glucosidases; inhibitors of membrane bound α-glucosidase; miglitol(Glyset®), and active derivatives, and analogs thereof; and acarbose(Precose®), and active derivative, and analogs thereof. Suitable agentsinclude long-alkyl-chain imino sugar derivatives that inhibit the HCVprotein p7. See, e.g., Pavlovic et al. (2003) Proc. Natl. Acad. Sci. USA100:6104-6108; and U.S. Patent Publication No. 2004/0110795. Suitableagents include, but are not limited to, deoxynojirimycin (DNJ),deoxygalactojirimycin (DGJ), N-butyl-deoxynojirimycin (NB-DNJ),N-nonyl-deoxynojirimycin (NN-DNJ), N-butyl-deoxygalactojirimycin(NB-DGJ), N-nonyl-deoxygalactojirimycin (NN-DGJ), NN-6deoxy-DGJ,N7-oxadecyl-DNJ, N7-oxanony-6deoxy-DGJ, perbutylated-N-butyl- 1-deoxynojiromycin (p-N-butyl-DNJ), and 6-O-butanoyl castanospermine.

Type I Interferon Receptor Agonists

In some embodiments, a subject method involves administration ofeffective amounts of an α-glucosidase inhibitor; and Type I interferonreceptor agonist. Type I interferon receptor agonists include an IFN-α;an IFN-β; an IFN-tau; an IFN-ω; antibody agonists specific for a Type Iinterferon receptor; and any other agonist of Type I interferonreceptor, including non-polypeptide agonists.

Interferon-Alpha

Any known IFN-α can be used in the instant invention. The term“interferon-alpha” as used herein refers to a family of relatedpolypeptides that inhibit viral replication and cellular proliferationand modulate immune response. The term “IFN-α” includes naturallyoccurring IFN-α; synthetic IFN-α; derivatized IFN-α (e.g., PEGylatedIFN-α, glycosylated IFN-α, and the like); and analogs of naturallyoccurring or synthetic IFN-α; essentially any IFN-α that has antiviralproperties, as described for naturally occurring IFN-α.

Suitable alpha interferons include, but are not limited to,naturally-occurring IFN-α (including, but not limited to, naturallyoccurring IFN-α2a, IFN-α2b); recombinant interferon alpha-2b such asIntron-A Interferon available from Schering Corporation, Kenilworth,N.J.; recombinant interferon alpha-2a such as Roferon interferonavailable from Hoffmann-La. Roche, Nutley, N. J.; recombinant interferonalpha-2C such as Berofor alpha 2 interferon available from BoehringerIngelheim Pharmaceutical, Inc., Ridgefield, Conn.; interferon alpha-n1,a purified blend of natural alpha interferons such as Sumiferonavailable from Sumitomo, Japan or as Wellferon interferon alpha-n1 (INS)available from the Glaxo-Wellcome Ltd., London, Great Britain; andinterferon alpha-n3 a mixture of natural alpha interferons made byInterferon Sciences and available from the Purdue Frederick Co.,Norwalk, Conn., under the Alferon Tradename.

The term “IFN-α” also encompasses consensus IFN-α. Consensus IFN-α (alsoreferred to as “CIFN” and “IFN-con” and “consensus interferon”)encompasses but is not limited to the amino acid sequences designated.IFN-con₁, IFN-con_(e) and IFN-con₃ which are disclosed in U.S. Pat. Nos.4,695,623 and 4,897,471; and consensus interferon as defined bydetermination of a consensus sequence of naturally occurring interferonalphas (e.g., Infergen®, InterMune, Inc., Brisbane, Calif.). IFN-con₁ isthe consensus interferon agent in the Infergen® alfacon-1 product. TheInfergen® consensus interferon product is referred to herein by itsbrand name (Infergen®) or by its generic name (interferon alfacon-1).DNA sequences encoding IFN-con may be synthesized as described in theaforementioned patents or other standard methods. Use of CIFN is ofparticular interest.

Also suitable for use in the present invention are fusion polypeptidescomprising an IFN-α and a heterologous polypeptide. Suitable IFN-αfusion polypeptides include, but are not limited to, Albuferon-alpha™ (afusion product of human albumin and IFN-α; Human Genome Sciences; see,e.g., Osborn et al. (2002) J. Pharmacol. Exp. Therap. 303:540-548). Alsosuitable for use in the present invention are gene-shuffled forms ofIFN-α. See., e.g., Masci et al. (2003) Curr. Oncol. Rep. 5:108-113.

PEGylated Interferon-Alpha

The term “IFN-α” also encompasses derivatives of IFN-α that arederivatized (e.g., are chemically modified) to alter certain propertiessuch as serum half-life. As such, the term “IFN-α” includes glycosylatedIFN-α; IFN-α derivatized with polyethylene glycol (“PEGylated IFN-α”);and the like. PEGylated IFN-α, and methods for making same, is discussedin, e.g., U.S. Pat. Nos. 5,382,657; 5,981,709; and 5,951,974. PEGylatedIFN-α encompasses conjugates of PEG and any of the above-described IFN-αmolecules, including, but not limited to, PEG conjugated to interferonalpha-2a (Roferon, Hoffman La-Roche, Nutley, N.J.), interferon alpha 2b(Intron, Schering-Plough, Madison, N.J.), interferon alpha-2c (BeroforAlpha, Boehringer Ingelheim, Ingelheim, Germany); and consensusinterferon as defined by determination of a consensus sequence ofnaturally occurring interferon alphas (Infergen®, InterMune, Inc.,Brisbane, Calif.).

Any of the above-mentioned IFN-α polypeptides can be modified with oneor more polyethylene glycol moieties, i.e., PEGylated. The PEG moleculeof a PEGylated IFN-α polypeptide is conjugated to one or more amino acidside chains of the IFN-α polypeptide. In some embodiments, the PEGylatedIFN-α contains a PEG moiety on only one amino acid. In otherembodiments, the PEGylated IFN-α contains a PEG moiety on two or moreamino acids, e.g., the IFN-α contains a PEG moiety attached to two,three, four, five, six, seven, eight, nine, or ten different amino acidresidues.

IFN-α may be coupled directly to PEG (i.e., without a linking group)through an amino group, a sulfhydryl group, a hydroxyl group, or acarboxyl group.

In some embodiments, the PEGylated IFN-α is PEGylated at or near theamino terminus (N-terminus) of the IFN-α polypeptide, e.g., the PEGmoiety is conjugated to the IFN-α polypeptide at one or more amino acidresidues from amino acid 1 through amino acid 4, or from amino acid 5through about 10.

In other embodiments, the PEGylated IFN-α is PEGylated at one or moreamino acid residues from about 10 to about 28.

In other embodiments, the PEGylated IFN-α is PEGylated at or near thecarboxyl terminus (C-terminus) of the IFN-α polypeptide, e.g., at one ormore residues from amino acids 156-166, or from amino acids 150 to 155.

In other embodiments, the PEGylated IFN-α is PEGylated at one or moreamino acid residues at one or more residues from amino acids 100-114.

The polyethylene glycol derivatization of amino acid residues at or nearthe receptor-binding and/or active site domains of the MN-α protein candisrupt the functioning of these domains. In certain embodiments of theinvention, amino acids at which PEGylation is to be avoided includeamino acid residues from amino acid 30 to amino acid 40; and amino acidresidues from amino acid 113 to amino acid 149.

In some embodiments, PEG is attached to IFN-α via a linking group. Thelinking group is any biocompatible linking group, where “biocompatible”indicates that the compound or group is non-toxic and may be utilized invitro or in vivo without causing injury, sickness, disease, or death.PEG can be bonded to the linking group, for example, via an ether bond,an ester bond, a thiol bond or an amide bond. Suitable biocompatiblelinking groups include, but are not limited to, an ester group, an amidegroup, an imide group, a carbamate group, a carboxyl group, a hydroxylgroup, a carbohydrate, a succinimide group (including, for example,succinimidyl succinate (SS), succinimidyl propionate (SPA), succinimidylbutanoate (SBA), succinimidyl carboxymethylate (SCM), succinimidylsuccinamide (SSA) or N-hydroxy succinimide (NHS)), an epoxide group, anoxycarbonylimidazole group (including, for example, carbonyldimidazole(CDI)), a nitro phenyl group (including, for example, nitrophenylcarbonate (NPC) or trichlorophenyl carbonate (TPC)), a trysylate group,an aldehyde group, an isocyanate group, a vinylsulfone group, a tyrosinegroup, a cysteine group, a histidine group or a primary amine.

Methods for making succinimidyl propionate (SPA) and succinimidylbutanoate (SBA) ester-activated PEGs are described in U.S. Pat. No.5,672,662 (Harris, et al.) and WO 97/03106.

Methods for attaching a PEG to an IFN-α polypeptide are known in theart, and any known method can be used. See, for example, by Park et al,Anticancer Res., 1:373-376 (1981); Zaplipsky and Lee, PolyethyleneGlycol Chemistry: Biotechnical and Biomedical Applications, J. M.Harris, ed., Plenum Press, NY, Chapter 21 (1992); U.S. Pat. No.5,985,265; U.S. Pat. No. 5,672,662 (Harris, et al.) and WO 97/03106.

Pegylated IFN-α, and methods for making same, is discussed in, e.g.,U.S. Pat. Nos. 5,382,657; 5,981,709; 5,985,265; and 5,951,974. PegylatedIFN-α encompasses conjugates of PEG and any of the above-described IFN-αmolecules, including, but not limited to, PEG conjugated to interferonalpha-2a (Roferon, Hoffman LaRoche, Nutley, N.J.), where PEGylatedRoferon is known as Pegasys (Hoffman LaRoche); interferon alpha 2b(Intron, Schering-Plough, Madison, N.J.), where PEGylated Intron isknown as PEG-Intron (Schering-Plough); interferon alpha-2c (BeroforAlpha, Boehringer Ingelheim, Ingelheim, Germany); and consensusinterferon (CIFN) as defined by determination of a consensus sequence ofnaturally occurring interferon alphas (Infergen®, InterMune, Inc.,Brisbane, Calif.), where PEGylated CIFN is referred to as PEG-CIFN.

In many embodiments, the PEG is a monomethoxyPEG molecule that reactswith primary amine groups on the IFN-α polypeptide. Methods of modifyingpolypeptides with monomethoxy PEG via reductive alkylation are known inthe art. See, e.g., Chamow et al. (1994) Bioconj. Chem. 5:133-140.

In one non-limiting example, PEG is linked to IFN-α via an SPA linkinggroup.

SPA esters of PEG, and methods for making same, are described in U.S.Pat. No. 5,672,662. SPA linkages provide for linkage to free aminegroups on the IFN-α polypeptide.

For example, a PEG molecule is covalently attached via a linkage thatcomprises an amide bond between a propionyl group of the PEG moiety andthe epsilon amino group of a surface-exposed lysine residue in the IFN-αpolypeptide. Such a bond can be formed, e.g., by condensation of anα-methoxy, omega propanoic acid activated ester of PEG (mPEGspa).

In some embodiments, the invention employs a PEG-modified CIFN, wherethe PEG moiety is attached to a lysine residue chosen from lys³¹, lys⁵⁰,lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, and lys¹⁶⁵. In these embodiments,the PEG moiety can be a linear PEG moiety having an average molecularweight of about 30 kD.

In other embodiments, the invention employs a PEG-modified CIFN, wherethe PEG moiety is attached to a lysine residue chosen from lys¹²¹,lys¹³⁴, lys¹³⁵, and lys¹⁶⁵. In these embodiments, the PEG moiety can bea linear PEG moiety having an average molecular weight of about 30 kD.

As one non-limiting example, one monopegylated CIFN conjugate preferredfor use herein has a linear PEG moiety of about 30 kD attached via acovalent linkage to the CIFN polypeptide, where the covalent linkage isan amide bond between a propionyl group of the PEG moiety and theepsilon amino group of a surface-exposed lysine residue in the CIFNpolypeptide, where the surface-exposed lysine residue is chosen fromlys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵,and the amide bond is formed by condensation of an α-methoxy, omegapropanoic acid activated ester of PEG.

Linking Groups

In some embodiments, PEG is attached to IFN-α via a linking group. Thelinking group is any biocompatible linking group, where “biocompatible”indicates that the compound or group is essentially non-toxic and may beutilized in vivo without causing a significant adverse response in thesubject, e.g., injury, sickness, disease, undesirable immune response,or death. PEG can be bonded to the linking group, for example, via anether bond, an ester bond, a thio ether bond or an amide bond. Suitablebiocompatible linking groups include, but are not limited to, an estergroup, an amide group, an imide group, a carbamate group, a carboxylgroup, a hydroxyl group, a carbohydrate, a succinimide group (including,for example, succinimidyl succinate (SS), succinimidyl propionate (SPA),succinimidyl butanoic acid (SBA), succinimidyl carboxymethylate (SCM),succinimidyl succinamide (SSA.) or N-hydroxy succinimide (NHS)), anepoxide group, an oxycarbonylimidazole group (including, for example,carbonyldimidazole (CDI)), a nitro phenyl group (including, for example,nitrophenyl carbonate (NPC) or trichlorophenyl carbonate (TPC)), atrysylate group, an aldehyde group, an isocyanate group, a vinylsulfonegroup, a tyrosine group, a cysteine group, a histidine group or aprimary amine.

In many embodiments, the PEG is a monomethoxyPEG molecule that reactswith primary amine groups on the IFN-α polypeptide. Methods of modifyingpolypeptides with monomethoxy PEG via reductive alkylation are known inthe art. See, e.g., Chamow et al. (1994) Bioconj. Chem. 5:133-140.

In one non-limiting example, PEG is linked to IFN-α via an SPA linkinggroup.

SPA esters of PEG, and methods for making same, are described in U.S.Pat. No. 5,672,662. SPA linkages provide for linkage to free aminegroups on the IFN-α polypeptide.

For example, a PEG molecule is covalently attached via a linkage thatcomprises an amide bond between a propionyl group of the PEG moiety andthe epsilon amino group of a surface-exposed lysine residue in the IFN-αpolypeptide. Such a bond can be formed, e.g., by condensation of anα-methoxy, omega propanoic acid activated ester of PEG (mPEGspa).

As one non-limiting example, monopegylated CIFN has a linear PEG moietyof about 30 kD attached via a covalent linkage to the CIFN polypeptide,where the covalent linkage is an amide bond between a propionyl group ofthe PEG moiety and the epsilon amino group of a surface-exposed lysineresidue in the CIFN polypeptide, where the surface-exposed lysineresidue is chosen from lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵, and the amidebond is formed by condensation of an ═-methoxy, omega propanoic acidactivated ester of PEG.

Methods for attaching a PEG molecule to an IFN-α polypeptide are knownin the art, and any known method can be used. See, for example, by Parket al, Anticancer Res., 1:373-376 (1981); Zaplipsky and Lee,Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications,J. M. Harris, ed., Plenum Press, NY, Chapter 21 (1992); and U.S. Pat.No. 5,985,265.

Polyethylene Glycol

Polyethylene glycol suitable for conjugation to an IFN-α polypeptide issoluble in water at room temperature, and has the general formulaR(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective group such asan alkyl or an alkanol group, and where n is an integer from 1 to 1000.Where R is a protective group, it generally has from 1 to 8 carbons.

In many embodiments, PEG has at least one hydroxyl group, e.g., aterminal hydroxyl group, which hydroxyl group is modified to generate afunctional group that is reactive with an amino group, e.g., an epsilonamino group of a lysine residue, a free amino group at the N-terminus ofa polypeptide, or any other amino group such as an amino group ofasparagine, glutamine, arginine, or histidine.

In other embodiments, PEG is derivatized so that it is reactive withfree carboxyl groups in the IFN-α polypeptide, e.g., the free carboxylgroup at the carboxyl terminus of the IFN-α polypeptide. Suitablederivatives of PEG that are reactive with the free carboxyl group at thecarboxyl-terminus of IFN-α include, but are not limited to PEG-amine,and hydrazine derivatives of PEG (e.g., PEG-NH—NH₂).

In other embodiments, PEG is derivatized such that it comprises aterminal thiocarboxylic acid group, -COSH, which selectively reacts withamino groups to generate amide derivatives. Because of the reactivenature of the thio acid, selectivity of certain amino groups over othersis achieved. For example, —SH exhibits sufficient leaving group abilityin reaction with N-terminal amino group at appropriate pH conditionssuch that the ε-amino groups in lysine residues are protonated andremain non-nucleophilic. On the other hand, reactions under suitable pHconditions may make some of the accessible lysine residues to react withselectivity.

In other embodiments, the PEG comprises a reactive ester such as anN-hydroxy succinimidate at the end of the PEG chain. Such anN-hydroxysuccinimidate-containing PEG molecule reacts with select aminogroups at particular pH conditions such as neutral 6.5-7.5. For example,the N-terminal amino groups may be selectively modified under neutral pHconditions. However, if the reactivity of the reagent were extreme,accessible —NH₂ groups of lysine may also react.

The PEG can be conjugated directly to the IFN-α polypeptide, or througha linker. In some embodiments, a linker is added to the IFN-αpolypeptide, forming a linker-modified IFN-α polypeptide. Such linkersprovide various functionalities, e.g., reactive groups such sulfhydryl,amino, or carboxyl groups to couple a PEG reagent to the linker-modifiedIFN-α polypeptide.

In some embodiments, the PEG conjugated to the IFN-α polypeptide islinear. In other embodiments, the PEG conjugated to the IFN-αpolypeptide is branched. Branched PEG derivatives such as thosedescribed in U.S. Pat. No. 5,643,575, “star-PEG's” and multi-armed PEG'ssuch as those described in Shearwater Polymers, Inc. catalog“Polyethylene Glycol Derivatives 1997-1998.” Star PEGs are described inthe art including, e.g., in U.S. Pat. No. 6,046,305.

PEG having a molecular weight in a range of from about 2 kDa to about100 kDa, is generally used, where the term “about,” in the context ofPEG, indicates that in preparations of polyethylene glycol, somemolecules will weigh more, some less, than the stated molecular weight.For example, PEG suitable for conjugation to IFN-α has a molecularweight of from about 2 kDa to about 5 kDa, from about 5 kDa to about 10kDa, from about 10 kDa to about 15 kDa, from about 15 kDa to about 20kDa, from about 20 kDa to about 25 kDa, from about 25 kDa to about 30kDa, from about 30 kDa to about 40 kDa, from about 40 kDa to about 50kDa, from about 50 kDa to about 60 kDa, from about 60 kDa to about 70kDa, from about 70 kDa to about 80 kDa, from about 80 kDa to about 90kDa, or from about 90 kDa to about 100 kDa. Preparing PEG-IFN-αconjugates

As discussed above, the PEG moiety can be attached, directly or via alinker, to an amino acid residue at or near the N-terminus, internally,or at or near the C-terminus of the IFN-α polypeptide. Conjugation canbe carried out in solution or in the solid phase.

N-Terminal Linkage

Methods for attaching a PEG moiety to an amino acid residue at or nearthe N-terminus of an IFN-α polypeptide are known in the art. See, e.g.,U.S. Pat. No. 5,985,265.

In some embodiments, known methods for selectively obtaining anN-terminally chemically modified IFN-α are used. For example, a methodof protein modification by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminus) available for derivatization in aparticular protein can be used. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved. Thereaction is performed at pH which allows one to take advantage of thepK_(a) differences between the ε-amino groups of the lysine residues andthat of the α-amino group of the N-terminal residue of the protein. Bysuch selective derivatization attachment of a PEG moiety to the IFN-α iscontrolled: the conjugation with the polymer takes place predominantlyat the N-terminus of the IFN-α and no significant modification of otherreactive groups, such as the lysine side chain amino groups, occurs.

C-Terminal Linkage

N-terminal-specific coupling procedures such as described in U.S. Pat.No. 5,985,265 provide predominantly monoPEGylated products. However, thepurification procedures aimed at removing the excess reagents and minormultiply PEGylated products remove the N-terminal blocked polypeptides.In terms of therapy, such processes lead to significant increases inmanufacturing costs. For example, examination of the structure of thewell-characterized Infergen® Alfacon-1 CIFN polypeptide amino acidsequence reveals that the clipping is approximate 5% at the carboxylterminus and thus there is only one major C-terminal sequence. Thus, insome embodiments, N-terminally PEGylated IFN-α is not used; instead, theIFN-α polypeptide is C-terminally PEGylated.

An effective synthetic as well as therapeutic approach to obtain monoPEGylated Infergen product is therefore envisioned as follows:

A PEG reagent that is selective for the C-terminal can be prepared withor without spacers. For example, polyethylene glycol modified as methylether at one end and having an amino function at the other end may beused as the starting material.

Preparing or obtaining a water-soluble carbodiimide as the condensingagent can be carried out. Coupling IFN-α (e.g., Infergen® Alfacon-1 CIFNor consensus interferon) with a water-soluble carbodiimide as thecondensing reagent is generally carried out in aqueous medium with asuitable buffer system at an optimal pH to effect the amide linkage. Ahigh molecular weight PEG can be added to the protein covalently toincrease the molecular weight.

The reagents selected will depend on process optimization studies. Anon-limiting example of a suitable reagent is EDAC or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The water solubility of EDAC allowsfor direct addition to a reaction without the need for prior organicsolvent dissolution. Excess reagent and the isourea formed as theby-product of the cross-linking reaction are both water-soluble and mayeasily be removed by dialysis or gel filtration. A concentrated solutionof EDAC in water is prepared to facilitate the addition of a small molaramount to the reaction. The stock solution is prepared and usedimmediately in view of the water labile nature of the reagent. Most ofthe synthetic protocols in literature suggest the optimal reactionmedium to be in pH range between 4.7 and 6.0. However the condensationreactions do proceed without significant losses in yields up to pH 7.5.Water may be used as solvent. In view of the contemplated use ofInfergen, preferably the medium will be 2-(N-morpholino)ethane sulfonicacid buffer pre-titrated to pH between 4.7 and 6.0. However, 0.1Mphosphate in the pH 7-7.5 may also be used in view of the fact that theproduct is in the same buffer. The ratios of PEG amine to the IFN-αmolecule is optimized such that the C-terminal carboxyl residue(s) areselectively PEGylated to yield monoPEGylated derivative(s).

Even though the use of PEG amine has been mentioned above by name orstructure, such derivatives are meant to be exemplary only, and othergroups such as hydrazine derivatives as in PEG-NH—NH₂ which will alsocondense with the carboxyl group of the IFN-α protein, can also be used.In addition to aqueous phase, the reactions can also be conducted onsolid phase. Polyethylene glycol can be selected from list of compoundsof molecular weight ranging from 300-40000. The choice of the variouspolyethylene glycols will also be dictated by the coupling efficiencyand the biological performance of the purified derivative in vitro andin vivo i.e., circulation times, anti viral activities etc.

Additionally, suitable spacers can be added to the C-terminal of theprotein. The spacers may have reactive groups such as SH, NH_(S;) orCOOH to couple with appropriate PEG reagent to provide the highmolecular weight IFN-α derivatives. A combined solid/solution phasemethodology can be devised for the preparation of C-terminal pegylatedinterferons. For example, the C-terminus of IFN-α is extended on a solidphase using a Gly-Gly-Cys-NH₂ spacer and then monopegylated in solutionusing activated dithiopyridyl-PEG reagent of appropriate molecularweights. Since the coupling at the C-terminus is independent of theblocking at the N-terminus, the envisioned processes and products willbe beneficial with respect to cost (a third of the protein is not wastedas in N-terminal PEGylation methods) and contribute to the economy ofthe therapy to treat chronic hepatitis C infections, liver fibrosis etc.

There may be a more reactive carboxyl group of amino acid residueselsewhere in the molecule to react with the PEG reagent and lead tomonoPEGylation at that site or lead to multiple PEGylations in additionto the —COOH group at the C-terminus of the IFN-α. It is envisioned thatthese reactions will be minimal at best owing to the steric freedom atthe C-terminal end of the molecule and the steric hindrance imposed bythe carbodiimides and the PEG reagents such as in branched chainmolecules. It is therefore the preferred mode of PEG modification forInfergen and similar such proteins, native or expressed in a hostsystem, which may have blocked N-termini to varying degrees to improveefficiencies and maintain higher in vivo biological activity.

Another method of achieving C-terminal PEGylation is as follows.Selectivity of C-terminal PEGylation is achieved with a stericallyhindered reagent which excludes reactions at carboxyl residues eitherburied in the helices or internally in IFN-α. For example, one suchreagent could be a branched chain PEG ˜40 kd in molecular weight andthis agent could be synthesized as follows:

OH₃C—(CH₂CH₂O)n-CH₂CH₂NH₂+Glutamic Acid i.e., HOCO—CH₂CH₂CH(NH2)—COOH iscondensed with a suitable agent e.g., dicyclohexyl carbodiimide orwater-soluble EDAC to provide the branched chain PEG agentOH₃C—(CH₂CH₂O)_(n)—CH₂CH₂NHCOCH(NH₂)CH₂OCH₃—(CH₂CH₂O)—CH₂CH₂.

This reagent can be used in excess to couple the amino group with thefree and flexible carboxyl group of IFN-α to form the peptide bond.

If desired, PEGylated IFN-α is separated from unPEGylated IFN-α usingany known method, including, but not limited to, ion exchangechromatography, size exclusion chromatography, and combinations thereofFor example, where the PEG-IFN-α conjugate is a monoPEGylated IFN-α, theproducts are first separated by ion exchange chromatography to obtainmaterial having a charge characteristic of monoPEGylated material (othermulti-PEGylated material having the same apparent charge may bepresent), and then the monoPEGylated materials are separated using sizeexclusion chromatography.

MonoPEG (30 kD, Linear)-ylated IFN-α

PEGylated IFN-α that is suitable for use in the present inventionincludes a monopegylated consensus interferon (CIFN) molecule comprisedof a single CIFN polypeptide and a single polyethylene glycol (PEG)moiety, where the PEG moiety is linear and about 30 kD in molecularweight and is directly or indirectly linked through a stable covalentlinkage to either the N-terminal residue in the CIFN polypeptide or alysine residue in the CIFN polypeptide. In some embodiments, the monoPEG(3.0 kD, linear)-ylated IFN-α is monoPEG (30 kD, linear)-ylatedconsensus IFN-α.

In some embodiments, the PEG moiety is linked to either the alpha-aminogroup of the N-term final residue in the CIFN polypeptide or theepsilon-amino group of a lysine residue in the CIFN polypeptide. Infurther embodiments, the linkage comprises an amide bond between the PEGmoiety and either the alpha-amino group of the N-terminal residue or theepsilon-amino group of the lysine residue in the CIFN polypeptide. Instill further embodiments, the linkage comprises an amide bond between apropionyl group of the PEG moiety and either the alpha-amino group ofthe N-terminal residue or the epsilon-amino group of the lysine residuein the CIFN polypeptide. In additional embodiments, the amide bond isformed by condensation of an alpha-methoxy, omega-propanoic acidactivated ester of the PEG moiety and either the alpha-amino group ofthe N-terminal residue or the epsilon-amino group of the lysine residuein the CIFN polypeptide, thereby forming a hydrolytically stable linkagebetween the PEG moiety and the CIFN polypeptide.

In some embodiments, the PEG moiety is linked to the N-terminal residuein the CIFN polypeptide. In other embodiments, the PEG moiety is linkedto the alpha-amino group of the N-terminal residue in the CIFNpolypeptide. In further embodiments, the linkage comprises an amide bondbetween the PEG moiety and the alpha-amino group of the N-terminalresidue in the CIFN polypeptide. In still further embodiments, thelinkage comprises an amide bond between a propionyl group of the PEGmoiety and the alpha-amino group of the N-terminal residue in the CIFNpolypeptide. In additional embodiments, the amide bond is formed bycondensation of an alpha-methoxy, omega-propanoic acid activated esterof the PEG moiety and the alpha-amino group of the N-terminal residue ofthe CIFN polypeptide.

In some embodiments, the PEG moiety is linked to a lysine residue in theCIFN polypeptide. In other embodiments, the PEG moiety is linked to theepsilon-amino group of a lysine residue in the CIFN polypeptide. Infurther embodiments, the linkage comprises an amide bond between the PEGmoiety and the epsilon-amino group of the lysine group in the CIFNpolypeptide. In still further embodiments, the linkage comprises anamide bond between a propionyl group of the PEG moiety and theepsilon-amino group of the lysine group in the CIFN polypeptide. Inadditional embodiments, the amide bond is formed by condensation of analpha-methoxy, omega-propanoic acid activated ester of the PEG moietyand the epsilon-amino group of the lysine residue in the CIFNpolypeptide.

In some embodiments, the PEG moiety is linked to a surface-exposedlysine residue in the CIFN polypeptide. In other embodiments, the PEGmoiety is linked to the epsilon-amino group of a surface-exposed lysineresidue in the CIFN polypeptide. In further embodiments, the linkagecomprises an amide bond between the PEG moiety and the epsilon-aminogroup of the surface-exposed lysine residue in the CIFN polypeptide. Instill further embodiments, the linkage comprises an amide bond between apropionyl group of the PEG moiety and the epsilon-amino group of thesurface-exposed lysine residue in the CIFN polypeptide. In additionalembodiments, the amide bond is formed by condensation of analpha-methoxy, omega-propanoic acid activated ester of the PEG moietyand the epsilon-amino group of the surface-exposed lysine residue in theCIFN polypeptide.

In some embodiments, the PEG moiety is linked to a lysine chosen fromlys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹³⁵, and ;ys¹⁶⁵ of the CIFN polypeptide.In other embodiments, the PEG moiety is linked to the epsilon-aminogroup of a lysine chosen from lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹³⁵, andlys¹⁶⁵ of the CIFN polypeptide. In other polypeptide. In furtherembodiments, the linkage comprises an amide bond between the PEG moietyand the epsilon-amino group of the chosen lysine residue in the CIFNpolypeptide. In still further embodiments, the linkage comprises anamide bond between a propionyl group of the PEG moiety and theepsilon-amino group of the chosen lysine residue in the CIFNpolypeptide. In additional embodiments, the amide bond is formed bycondensation of an alpha-methoxy, omega-propanoic acid activated esterof the PEG moiety and the epsilon-amino group of the chosen lysineresidue in the CIFN polypeptide.

In some embodiments, the PEG moiety is linked to a lysine chosen fromlys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide. In otherembodiments, the PEG moiety is linked to the epsilon-amino group of alysine chosen from lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFNpolypeptide. In further embodiments, the linkage comprises an amide bondbetween the PEG moiety and the epsilon-amino group of the chosen lysineresidue in the CIFN polypeptide. In still further embodiments, thelinkage comprises an amide bond between a propionyl group of the PEGmoiety and the epsilon-amino group of the chosen lysine residue in theCIFN polypeptide. In additional embodiments, the amide bond is formed bycondensation of an alpha-methoxy, omega-propanoic acid activated esterof the PEG moiety and the epsilon-amino group of the chosen lysineresidue in the CIFN polypeptide.

In connection with the above-described monopegylated CIFN molecules, theinvention contemplates use of embodiments of each such molecule wherethe CIFN polypeptide is chosen from interferon alpha-con₁, interferonalpha-con₂, and interferon alpha-con₃, the amino acid sequences of whichCIFN polypeptides are disclosed in U.S. Pat. No. 4,695,623.

Populations of IFN-α

In addition, any of the methods of the invention can employ a PEGylatedIFN-α composition that comprises a population of monopegylated IFNamolecules, where the population consists of one or more species ofmonopegylated IFNa molecules as described above. Thus, in someembodiments, the composition comprises a population of modified IFN-αpolypeptides, each with a single PEG molecule linked to a single aminoacid residue of the polypeptide.

In some of these embodiments, the population comprises a mixture of afirst IFN-α polypeptide linked to a PEG molecule at a first amino acidresidue; and at least a second IFN-α polypeptide linked to a PEGmolecule at a second amino acid residue, wherein the first and secondIFN-α polypeptides are the same or different, and wherein the locationof the first amino acid residue in the amino acid sequence of the firstIFN-α polypeptide is not the same as the location of the second aminoacid residue in the second IFN-α polypeptide. As one non-limitingexample, an IFN-α composition comprises a population of PEG-modifiedIFN-α polypeptides, the population comprising an IFN-α polypeptidelinked at its amino terminus to a linear PEG molecule; and an IFN-αpolypeptide linked to a linear PEG molecule at a lysine residue.

Generally, a given modified IFN-α species represents from about 0.5% toabout 99.5% of the total population of monopegylated IFNa polypeptidemolecules in a population, e.g, a given modified IFN-α speciesrepresents about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%,about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, orabout 99.5% of the total population of monopegylated IFN-α polypeptidemolecules in a population. In some embodiments, an IFN-α compositioncomprises a population of monopegylated IFN-α polypeptides, whichpopulation comprises at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, or at least about 99%, IFN-α polypeptideslinked to PEG at the same site, e.g., at the N-terminal amino acid.

In particular embodiments of interest, an IFN-α composition comprises apopulation of monopegylated CIFN molecules, the population consisting ofone or more species of molecules, where each species of molecules ischaracterized by a single CIFN polypeptide linked, directly orindirectly in a covalent linkage, to a single linear PEG moiety of about30 kD in molecular weight, and where the linkage is to either a lysineresidue in the CIFN polypeptide, or the N-terminal amino acid residue ofthe CIFN polypeptide.

The amino acid residue to which the PEG is attached is in manyembodiments the N-terminal amino acid residue. In other embodiments, thePEG moiety is attached (directly or via a linker) to a surface-exposedlysine residue. In additional embodiments, the PEG moiety is attached(directly or via a linker) to a lysine residue chosen from lys³¹, lys⁵⁰,lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFNpolypeptide. In further embodiments, the PEG moiety is attached(directly or via a linker) to a lysine residue chosen from lys¹²¹,1ys¹³⁴, lys¹³⁵, and lys¹⁶⁵ of the CIFN polypeptide.

As an example, in some embodiments, an I FN-α composition comprises apopulation of monopegylated CIFN molecules, consisting of a firstmonopegylated CIFN polypeptide species of molecules characterized by aPEG moiety linked at the N-terminal amino acid residue of a first CIFNpolypeptide, and a second monopegylated CIFN polypeptide species ofmolecules characterized by a PEG moiety linked to a first lysine residueof a second CIFN polypeptide, where the first and second CIFNpolypeptides are the same or different. An IFN-α composition can furthercomprise at least one additional monopegylated CIFN polypeptide speciesof molecules characterized by a PEG moiety linked to a lysine residue inthe CIFN polypeptide, where the location of the linkage site in eachadditional monopegylated CIFN polypeptide species is not the same as thelocation of the linkage site in any other species. In all species inthis example, the PEG moiety is a linear PEG moiety having an averagemolecular weight of about 30 kD.

As another example, in some embodiments, an IFN-α composition comprisesa population of monopegylated CIFN molecules, consisting of a firstmonopegylated CIFN polypeptide species of molecules characterized by aPEG moiety linked at the N-terminal amino acid residue of a first CIFNpolypeptide, and a second monopegylated CIFN polypeptide species ofmolecules characterized by a PEG moiety linked to a firstsurface-exposed lysine residue of a second CIFN polypeptide, where thefirst and second CIFN polypeptides are the same or different. An IFN-αcomposition can further comprise at least one additional monopegylatedCIFN polypeptide species of molecules characterized by a PEG moietylinked to a surface-exposed lysine residue in the CIFN polypeptide,where the location of the linkage site in each additional monopegylatedCIFN polypeptide species is not the same as the location of the linkagesite in any other species. In all species in this example, the PEGmoiety is a linear PEG moiety having an average molecular weight ofabout 30 kD.

As another example, in some embodiments, an IFN-α composition comprisesa population of monopegylated CIFN molecules, consisting of a firstmonopegylated CIFN polypeptide species of molecules characterized by aPEG moiety linked at the N-terminal amino acid residue of a first CIFNpolypeptide, and a second monopegylated CIFN polypeptide species ofmolecules characterized by a PEG moiety linked to a first lysine residueselected from one of lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴,lys¹³⁵, and lys¹⁶⁵ in a second CIFN polypeptide, where the first andsecond CIFN polypeptides are the same or different. An IFN-α compositioncan further comprise a third monopegylated CIFN polypeptide species ofmolecules characterized by a PEG moiety linked to a second lysineresidue selected from one of lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²²,lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a third CIFN polypeptide, where the thirdCIFN polypeptide is the same or different from either of the first andsecond CIFN polypeptides, where the second lysine residue is located ina position in the amino acid sequence of the third CIFN polypeptide thatis not the same as the position of the first lysine residue in the aminoacid sequence of the second CIFN polypeptide. A composition suitable foruse in a subject method may further comprise at least one additionalmonopegylated CIFN polypeptide species of molecules characterized by aPEG moiety linked to one of lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²²,lys¹³⁴, lys¹³⁵, and lys¹⁶⁵, where the location of the linkage site ineach additional monopegylated CIFN polypeptide species is not the sameas the location of the linkage site in any other species. In all speciesin this example, the PEG moiety is a linear PEG moiety having an averagemolecular weight of about 30 kD.

As another example, in some embodiments, an IFN-α composition comprisesa population of monopegylated CIFN molecules, consisting of a firstmonopegylated CIFN polypeptide species of molecules characterized by aPEG moiety linked at the N-terminal amino acid residue of a first CIFNpolypeptide, and a second monopegylated CIFN polypeptide species ofmolecules characterized by a PEG moiety linked to a first lysine residueselected from one of lys^(121, lys) ¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a secondCIFN polypeptide, where the first and second CIFN polypeptides are thesame or different. A composition suitable for use in a subject methodcan further comprise a third monopegylated CIFN polypeptide species ofmolecules characterized by a PEG moiety linked to a second lysineresidue selected from one of lys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in athird CIFN polypeptide, where the third CIFN polypeptide is the same ordifferent from either of the first and second CIFN polypeptides, wherethe second lysine residue is located in a position in the amino acidsequence of the third CIFN polypeptide that is not the same as theposition of the first lysine residue in the amino acid sequence of thesecond CIFN polypeptide. An IFN-α composition may further comprise atleast one additional monopegylated CIFN polypeptide species of moleculescharacterized by a PEG moiety linked to one of lys¹²¹, lys¹³⁴, lys¹³⁵,and lys¹⁶⁵, where the location of the linkage site in each additionalmonopegylated CIFN polypeptide species is not the same as the locationof the linkage site in any other species. In all species in thisexample, the PEG moiety is a linear PEG moiety having an averagemolecular weight of about 30 kD.

As another non-limiting example, in some embodiments, a compositionsuitable for use in a subject method comprises a population ofmonopegylated CIFN molecules, consisting of a first monopegylated CIFNpolypeptide species of molecules characterized by a PEG moiety linked toa first lysine residue in a first CIFN polypeptide; and a secondmonopegylated CIFN polypeptide species of molecules characterized by aPEG moiety linked at a second lysine residue in a second CIFNpolypeptide, where the first and second CIFN polypeptides are the sameor different, and where the first lysine is located in a position in theamino acid sequence of the first CIFN polypeptide that is not the sameas the position of the second lysine residue in the amino acid sequenceof the second CIFN polypeptide. An IFN-α composition may furthercomprise at least one additional monopegylated CIFN species of moleculescharacterized by a PEG moiety linked to a lysine residue in the CIFNpolypeptide, where the location of the linkage site in each additionalmonopegylated CIFN polypeptide species is not the same as the locationof the linkage site in any other species. In all species in thisexample, the PEG moiety is a linear PEG moiety having an averagemolecular weight of about 30 kD.

As another non-limiting example, in some embodiments, an IFN-αcomposition suitable for use in a subject method comprises a populationof monopegylated CIFN molecules, consisting of a first monopegylatedCIFN polypeptide species of molecules characterized by a PEG moietylinked at a first lysine residue chosen from lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴,lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵ in a first CIFN polypeptide;and a second monopegylated CIFN polypeptide species of moleculescharacterized by a PEG moiety linked at a second lysine residue chosenfrom lys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, andlys¹⁶⁵ in a second CIFN polypeptide, where the first and second CIFNpolypeptides are the same or different, and where the second lysineresidue is located in a position in the amino acid sequence of thesecond CIFN polypeptide that is not the same as the position of thefirst lysine residue in the first CIFN polypeptide. The composition mayfurther comprise at least one additional monopegylated CIFN polypeptidespecies of molecules characterized by a PEG moiety linked to one of1ys³¹, lys⁵⁰, lys⁷¹, lys⁸⁴, lys¹²¹, lys¹²², lys¹³⁴, lys¹³⁵, and lys¹⁶⁵,where the location of linkage site in each additional monopegylated CIFNpolypeptide species is not the same as the location of the linkage sitein any other species. In all species in this example, the PEG moiety isa linear PEG moiety having an average molecular weight of about 30 kD.

As another non-limiting example, in some embodiments, an IFN-αcomposition suitable for use in a subject method comprises a populationof monopegylated CIFN molecules, consisting of a first monopegylatedCIFN polypeptide species of molecules characterized by a PEG moietylinked at a first lysine residue chosen from lys¹²¹, lys¹³⁴, lys¹³⁵, andlys¹⁶⁵ in a first CIFN polypeptide; and a second monopegylated CIFNpolypeptide species of molecules characterized by a PEG moiety linked ata second lysine residue chosen from lys^(121, lys) ¹³⁴, lys¹³⁵, andlys¹⁶⁵ in a second CIFN polypeptide, where the first and second CIFNpolypeptides are the same or different, and where the second lysineresidue is located in a position in the amino acid sequence of thesecond CIFN polypeptide that is not the same as the position of thefirst lysine residue in the first CIFN polypeptide. The composition mayfurther comprise at least one additional monopegylated CIFN polypeptidespecies of molecules characterized by a PEG moiety linked to one oflys¹²¹, lys¹³⁴, lys¹³⁵, and lys¹⁶⁵, where the location of the linkagesite in each additional monopegylated CIFN polypeptide species is notthe same as the location of the linkage site in any other species. Inall species in this example, the PEG moiety is a linear PEG moietyhaving an average molecular weight of about 30 kD.

As another non-limiting example, in some embodiments, an IFN-αcomposition suitable for use in a subject method comprises amonopegylated population of CIFN molecules, consisting of a firstmonopegylated CIFN polypeptide species of molecules characterized by aPEG moiety linked to a first surface-exposed lysine residue in a firstCIFN polypeptide; and a second monopegylated CIFN polypeptide species ofmolecules characterized by a PEG moiety linked at a secondsurface-exposed lysine residue in a second CIFN polypeptide, where thefirst and second CIFN polypeptides are the same or different, and wherethe first surface-exposed lysine is located in a position in the aminoacid sequence of the first CIFN polypeptide that is not the same as theposition of the second surface-exposed lysine residue in the amino acidsequence of the second CIFN polypeptide. An IFN-α composition mayfurther comprise at least one additional monopegylated CIFN species ofmolecules characterized by a PEG moiety linked to a surface-exposedlysine residue in the CIFN polypeptide, where the location of thelinkage site in each additional monopegylated CIFN polypeptide speciesis not the same as the location of the linkage site in any otherspecies. In all species in this example, the PEG moiety is a linear PEGmoiety having an average molecular weight of about 30 kD.

In connection with each of the above-described populations ofmonopegylated CIFN molecules, the invention contemplates use ofembodiments where the molecules in each such population comprise a CIFNpolypeptide chosen from interferon alpha-con₁, interferon alpha-con₂,and interferon alpha-con₃.

The invention further features use in a subject method of a product thatis produced by the process of reacting CIFN polypeptide with asuccinimidyl ester of alpha-methoxy, omega-propionylpoly(ethyleneglycol) (mPEGspa) that is linear and about 30 kD in molecular weight,where the reactants are initially present at a molar ratio of about 1:1to about 1:5 CIFN:mPEGspa, and where the reaction is conducted at a pHof about 7 to about 9, followed by recovery of the monopegylated CIFNproduct of the reaction. In one embodiment, the reactants are initiallypresent at a molar ratio of about 1:3 CIFN:mPEGspa and the reaction isconducted at a pH of about 8. In another embodiment where the modifiedIFN-α is generated by a scaled-up procedure needed for toxicological andclinical investigations, the reactants are initially present in a molarratio of 1:2 CIFN:mPEGspa and the reaction is conducted at a pH of about8.0.

In connection with the above-described product-by-process, the inventioncontemplates use of embodiments where the CIFN reactant is chosen frominterferon alpha-con₁, interferon alpha-con₂, and interferon alpha-con₃.

In some embodiments, the present invention contemplates use of a knownhyperglycosylated polypeptide variant of a parent protein therapeutic.In some embodiments, the parent protein therapeutic is an interferon,and a known hyperglycosylated polypeptide variant comprises (1) acarbohydrate moiety covalently attached to at least one non-nativeglycosylation site not found in the parent interferon and/or (2) acarbohydrate moiety covalently attached to at least one nativeglycosylation site found but not glycosylated in the parent interferon.

In some embodiments, the known hyperglycosylated polypeptide variant isany glycosylated synthetic Type I interferon receptor polypeptideagonist.

Suitable known hyperglycosylated polypeptide variants includehyperglycosylated forms of any parent alpha interferon polypeptide. Inone aspect, a known hyperglycosylated variant of a parent alphainterferon polypeptide has an amino acid sequence that differs from theamino acid sequence of the parent polypeptide to the extent that thevariant comprises one or more glycosylation sites not found in theparent polypeptide.

In another aspect, the parent polypeptide is IFN-α2a and the knownhyperglycosylated polypeptide variant is an [D99N]IFN-α2a glycopeptide,where the [D99N]IFN-α2a glycopeptide is a variant of IFN-α2a having (a)an asparagine residue in place of the native aspartic acid residue atamino acid position 99 in the amino acid sequence of IFN-α2a and (b) acarbohydrate moiety covalently attached to the R-group of saidasparagine residue.

In another aspect, the parent polypeptide is IFN-α2a and the knownhyperglycosylated polypeptide variant is an [D99N, D105N]IFN-α2aglycopeptide, where the [D99N, D105N]IFN-α2a glycopeptide is a variantof IFN-α2a having (a) an asparagine residue in place of the nativeaspartic acid residue at each of amino acid positions 99 and 105 in theamino acid sequence of IFN-α2a and (b) a carbohydrate moiety covalentlyattached to the R-group of each of said asparagine residues.

In another aspect, the parent polypeptide is IFN-α2b and the knownhyperglycosylated polypeptide variant is an [D99N]IFN-α2b glycopeptide,where the [D99N]IFN-α2b glycopeptide is a variant of IFN-α2b having (a)an asparagine residue in place of the native aspartic acid residue atamino acid position 99 in the amino acid sequence of IFN-α2b and (b) acarbohydrate moiety covalently attached to the R-group of saidasparagine residue.

In another aspect, the parent polypeptide is IFN-α2b and the knownhyperglycosylated polypeptide variant is an [D99N, D105N]IFN-α2bglycopeptide, where the [D99N, D105N]IFN-α2b glycopeptide is a variantof IFN-α2b having (a) an asparagine residue in place of the nativeaspartic acid residue at each of amino acid positions 99 and 105 in theamino acid sequence of IFN-α2b and (b) a carbohydrate moiety covalentlyattached to the R-group of each of said asparagine residues.

Suitable alpha interferons further include consensus IFN-α. ConsensusIFN-α (also referred to as “CIFN” and “IFN-con” and “consensusinterferon”) encompasses but is not limited to the amino acid sequencesdesignated IFN-con₁, IFN-con₂ and IFN-con₃ which are disclosed in U.S.Pat. Nos. 4,695,623 and 4,897,471; and consensus interferon as definedby determination of a consensus sequence of naturally occurringinterferon alphas (e.g., Infergen®, InterMune, Inc., Brisbane, Calif.).IFN-con₁ is the consensus interferon agent in the Infergen® alfacon-1product. The Infergen® consensus interferon product is referred toherein by its brand name (Infergen®) or by its generic name (interferonalfacon-1).

Suitable known hyperglycosylated polypeptide variants includehyperglycosylated forms of any parent consensus IFN-α polypeptide. Inone aspect, a known hyperglycosylated variant of a parent consensusIFN-α polypeptide has an amino acid sequence that differs from the aminoacid sequence of the parent polypeptide to the extent that the variantcomprises one or more glycosylation sites not found in a parentpolypeptide.

In another aspect, the parent polypeptide is the interferon alfacon-1polypeptide and the known hyperglycosylated polypeptide variant is an[D99N]interferon alfacon-1 glycopeptide, where the [D99N]interferonalfacon-1 glycopeptide is a variant of the interferon alfacon-1polypeptide having (a) an asparagine residue substituted for the nativeaspartic acid residue at amino acid position 99 in the amino acidsequence of Infergen (interferon alfacon-1) and (b) a carbohydratemoiety covalently attached to the R-group of said asparagine residue.

In another aspect, the parent polypeptide is the interferon alfacon-1polypeptide and the known hyperglycosylated polypeptide variant is an[D99N, D105N]interferon alfacon-1 glycopeptide, where the [D99N,D105N]interferon alfacon-1 glycopeptide is a variant of the interferonalfacon-1 polypeptide having (a) an asparagine residue substituted foreach of the native aspartic acid residues at amino acid positions 99 and105 in the amino acid sequence of Infergen and (b) a carbohydrate moietycovalently attached to the R-group of each of said asparagine residues.

In another aspect, the parent polypeptide is the interferon alfacon-1polypeptide and the known hyperglycosylated polypeptide variant is an[D99N, D105N, E134N]interferon alfacon-1 glycopeptide, where the [D99N,D105N, E134N]interferon alfacon-1 glycopeptide is a variant of theinterferon alfacon-1 polypeptide having (a) an asparagine residuesubstituted for each of the native aspartic acid, aspartic acid, andglutamic acid residues at amino acid positions 99, 105 and 134,respectively, in the amino acid sequence of Infergen and (b) acarbohydrate moiety covalently attached to the R-group of each of saidasparagine residues.

In another aspect, the parent polypeptide is the interferon alfacon-1polypeptide and the known hyperglycosylated polypeptide variant is an[D99N, E134N]interferon alfacon-1 glycopeptide, where the [D99N,E134N]interferon alfacon-1 glycopeptide is a variant of the interferonalfacon-1 polypeptide having (a) an asparagine residue substituted foreach of the native aspartic acid and glutamic acid residues at aminoacid positions 99 and 134, respectively, in the amino acid sequence ofInfergen and (b) a carbohydrate moiety covalently attached to theR-group of each of said asparagine residues.

In another aspect, the parent polypeptide is the interferon alfacon-1polypeptide and the known hyperglycosylated polypeptide variant is an[D105N, E134N]interferon alfacon-1 glycopeptide, where the [D105N,E134N]interferon alfacon-1 glycopeptide is a variant of the interferonalfacon-1 polypeptide having (a) an asparagine residue substituted foreach of the native aspartic acid and glutamic acid residues at aminoacid positions 105 and 134, respectively, in the amino acid sequence ofInfergen and (b) a carbohydrate moiety covalently attached to theR-group of each of said asparagine residues.

In another aspect, the parent polypeptide is the interferon alfacon-1polypeptide and the known hyperglycosylated polypeptide variant is an[D99N, D105N, E134T]interferon alfacon-1 glycopeptide, where the [D99N,D105N, E134T]interferon alfacon-1 glycopeptide is a variant of theinterferon alfacon-1 polypeptide having (a) an asparagine residuesubstituted for each of the native aspartic acid residues at amino acidpositions 99 and 105 in the amino acid sequence of Infergen (b) athreonine residue substituted for the native glutamic acid residue atamino acid position 134 in the amino acid sequence of Infergen and (c) acarbohydrate moiety covalently attached to the R-group of each of saidasparagine and threonine residues.

In another aspect, the parent polypeptide is the interferon alfacon-1polypeptide and the known hyperglycosylated polypeptide variant is an[D99N, E134T]interferon alfacon-1 glycopeptide, where the [D99N,E134T]interferon alfacon-1 glycopeptide is a variant of the interferonalfacon-1 polypeptide having (a) an asparagine residue substituted forthe native aspartic acid residue at amino acid position 99 in the aminoacid sequence of Infergen (b) a threonine residue substituted for thenative glutamic acid residue at amino acid position 134 in the aminoacid sequence of Infergen and (c) a carbohydrate moiety covalentlyattached to the R-group of each of said asparagine and threonineresidues.

In another aspect, the parent polypeptide is the interferon alfacon-1polypeptide and the known hyperglycosylated polypeptide variant is an[D105N, E134T]interferon alfacon-1 glycopeptide, where the [D105N,E134T]interferon alfacon-1 glycopeptide is a variant of the interferonalfacon-1 polypeptide having (a) an asparagine residue substituted forthe native aspartic acid residue at amino acid position 105 in the aminoacid sequence of Infergen (b) a threonine residue substituted for thenative glutamic acid residue at amino acid position 134 in the aminoacid sequence of Infergen and (c) a carbohydrate moiety covalentlyattached to the R-group of each of said asparagine and threonineresidues.

In another aspect, a known hyperglycosylated polypeptide variant of aparent interferon-alpha therapeutic differs from the parentinterferon-alpha therapeutic to the extent that the knownhyperglycosylated polypeptide variant comprises (1) a carbohydratemoiety covalently attached to a non-native glycosylation site not foundin the parent interferon-alpha therapeutic and/or (2) a carbohydratemoiety covalently attached to a native glycosylation site found but notglycosylated in the parent interferon-alpha therapeutic.

IFN-β

In some embodiments, the at least one additional therapeutic agent in asubject combination therapy includes an IFN-β. The term interferon-beta(“IFN-β”) includes IFN-β polypeptides that are naturally occurring;non-naturally-occurring IFN-β polypeptides; and analogs and variants ofnaturally occurring or non-naturally occurring IFN-β that retainantiviral activity of a parent naturally-occurring or non-naturallyoccurring IFN-β.

Any of a variety of beta interferons can be used in a subject treatmentmethod. Suitable beta interferons include, but are not limited to,naturally-occurring IFN-β; IFN-β1a, e.g., Avonex® (Biogen, Inc.), andRebif® (Serono, SA); IFN-β1b (Betaseron®; Berlex); and the like.

The IFN-β formulation may comprise an N-blocked species, wherein theN-terminal amino acid is acylated with an acyl group, such as a formylgroup, an acetyl group, a malonyl group, and the like. Also suitable foruse is a consensus IFN-β.

IFN-β polypeptides can be produced by any known method. DNA sequencesencoding IFN-β may be synthesized using standard methods. In manyembodiments, IFN-β polypeptides are the products of expression ofmanufactured DNA sequences transfomied or transfected into bacterialhosts, e.g., E. coli, or in eukaryotic host cells (e.g., yeast;mammalian cells, such as CHO cells; and the like). In these embodiments,the IFN-β is “recombinant IFN-β.” Where the host cell is a bacterialhost cell, the IFN-β is modified to comprise an N-terminal methionine.

It is to be understood that IFN-β as described herein may comprise oneor more modified amino acid residues, e.g., glycosylations, chemicalmodifications, and the like.

IFN-tau

In some embodiments, the at least one additional therapeutic agent in asubject combination therapy includes an IFN-tau. The term“interferon-tau” (IFN-tau) includes IFN-tau polypeptides that arenaturally occurring; non-naturally-occurring IFN-tau polypeptides; andanalogs and variants of naturally occurring or non-naturally occurringIFN-tau that retain antiviral activity of a parent naturally-occurringor non-naturally occurring IFN-tau.

Suitable tau interferons include, but are not limited to,naturally-occurring IFN-tau; Tauferon® (Pepgen Corp.); and the like.

IFN-tau may comprise an amino acid sequence as set forth in any one ofGenBank Accession Nos. P15696; P56828; P56832; P56829; P56831; Q29429;Q28595; Q28594; S08072; Q08071; Q08070; Q08053; P56830; P28169; P28172;and P28171. The sequence of any known IFN-tau polypeptide may be alteredin various ways known in the art to generate targeted changes insequence. A variant polypeptide will usually be substantially similar tothe sequences provided herein, i.e. will differ by at least one aminoacid, and may differ by at least two but not more than about ten aminoacids. The sequence changes may be substitutions, insertions ordeletions. Conservative amino acid substitutions typically includesubstitutions within the following groups: (glycine, alanine); (valine,isoleucine, leucine); (aspartic acid, glutamic acid); (asparagine,glutamine); (serine, threonine); (lysine, arginine); or (phenylalanine,tyrosine).

Modifications of interest that may or may not alter the primary aminoacid sequence include chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation; changes in amino acid sequence thatintroduce or remove a glycosylation site; changes in amino acid sequencethat make the protein susceptible to PEGylation; and the like. Alsoincluded are modifications of glycosylation, e.g. those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps; e.g. byexposing the polypeptide to enzymes that affect glycosylation, such asmammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

The IFN-tau formulation may comprise an N-blocked species, wherein theN-terminal amino acid is acylated with an acyl group, such as a formylgroup, an acetyl group, a malonyl group, and the like. Also suitable foruse is a consensus IFN-tau.

IFN-tau polypeptides can be produced by any known method. DNA sequencesencoding IFN-tau may be synthesized using standard methods. In manyembodiments, IFN-tau polypeptides are the products of expression ofmanufactured DNA sequences transformed or transfected into bacterialhosts, e.g., E. coli, or in eukaryotic host cells (e.g., yeast;mammalian cells, such as CHO cells; and the like). In these embodiments,the IFN-tau is “recombinant IFN-tau.” Where the host cell is a bacterialhost cell, the IFN-tau is modified to comprise an N-terminal methionine.

It is to be understood that IFN-tau as described herein may comprise oneor more modified amino acid residues, e.g., glycosylations, chemicalmodifications, and the like.

IFN-ω

In some embodiments, the at least one additional therapeutic agent in asubject combination therapy includes in IFN-omega. The terminterferon-omega (“IFN-ω”) includes IFN-ω polypeptides that arenaturally occurring; non-naturally-occurring IFN-ω polypeptides; andanalogs and variants of naturally occurring or non-naturally occurringIFN-ω that retain antiviral activity of a parent naturally-occurring ornon-naturally occurring IFN-ω.

Any known omega interferon can be used in a subject treatment method.Suitable IFN-ω include, but are not limited to, naturally-occurringIFN-ω; recombinant IFN-ω, e.g., Biomed 510 (BioMedicines); and the like.

IFN-ω may comprise an amino acid sequence as set forth in GenBankAccession No. NP_(—)002168; or AAA70091. The sequence of any known IFN-ωpolypeptide may be altered in various ways known in the art to generatetargeted changes in sequence. A variant polypeptide will usually besubstantially similar to the sequences provided herein, i.e. will differby at least one amino acid, and may differ by at least two but not morethan about ten amino acids. The sequence changes may be substitutions,insertions or deletions. Conservative amino acid substitutions typicallyinclude substitutions within the following groups: (glycine, alanine);(valine, isoleucine, leucine); (aspartic acid. glutamic acid);(asparagine, glutamine); (serine, threonine); (lysine, arginine); or(phenylalanine, tyrosine).

Modifications of interest that may or may not alter the primary aminoacid sequence include chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation; changes in amino acid sequence thatintroduce or remove a glycosylation site; changes in amino acid sequencethat make the protein susceptible to PEGylation; and the like. Alsoincluded are modifications of glycosylation, e.g. those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps; e.g. byexposing the polypeptide to enzymes that affect glycosylation, such asmammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

The IFN-ω formulation may comprise an N-blocked species, wherein theN-terminal amino acid is acylated with an acyl group, such as a formylgroup, an acetyl group, a malonyl group, and the like. Also suitable foruse is a consensus IFN-ω.

IFN-ω polypeptides can be produced by any known method. DNA sequencesencoding IFN-ω may be synthesized using standard methods. In manyembodiments, IFN-ω polypeptides are the products of expression ofmanufactured DNA sequences transformed or transfected into bacterialhosts, e.g., E. coli, or in eukaryotic host cells (e.g., yeast;mammalian cells, such as CHO cells; and the like). In these embodiments,the IFN-ω is “recombinant IFN-co.” Where the host cell is a bacterialhost cell, the IFN-ω is modified to comprise an N-terminal methionine.

It is to be understood that IFN-ω as described herein may comprise oneor more modified amino acid residues, e.g., glycosylations, chemicalmodifications, and the like.

Type III Interferon Receptor Agonists

In some embodiments, the at least one additional therapeutic agent in asubject combination therapy includes a Type III interferon receptoragonist. Type III interferon agonists include an IL-28b polypeptide; andIL-28a polypeptide; and IL-29 polypeptide; antibody specific for a TypeIII interferon receptor; and any other agonist of Type III interferonreceptor, including non-polypeptide agonists.IL-28A, IL-28B, and IL-29(referred to herein collectively as “Type III interferons” or “Type IIIIFNs”) are described in Sheppard et al. (2003) Nature 4:63-68. Eachpolypeptide binds a heterodimeric receptor consisting of IL-10 receptorβ chain and an IL-28 receptor α. Sheppard et al. (2003), supra. Theamino acid sequences of IL-28A, IL-28B, and IL-29 are found underGenBank Accession Nos. NP_(—)742150, NP_(—)742151, and NP_(—)742152,respectively.

The amino acid sequence of a Type III IFN polypeptide may be altered invarious ways known in the art to generate targeted changes in sequence.A variant polypeptide will usually be substantially similar to thesequences provided herein, i.e. will differ by at least one amino acid,and may differ by at least two but not more than about ten amino acids.The sequence changes may be substitutions insertions or deletions.Scanning mutations that systematically introduce alanine, or otherresidues, may be used to determine key amino acids. Specific amino acidsubstitutions of interest include conservative and non-conservativechanges. Conservative amino acid substitutions typically includesubstitutions within the following groups: (glycine, alanine); (valine,isoleucine, leucine); (aspartic acid, glutamic acid); (asparagine,glutamine); (serine, threonine); (lysine, arginine); or (phenylalanine,tyrosine).

Modifications of interest that may or may not alter the primary aminoacid sequence include chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation; changes in amino acid sequence thatintroduce or remove a glycosylation site; changes in amino acid sequencethat make the protein susceptible to PEGylation; and the like. Alsoincluded are modifications of glycosylation, e.g. those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps; e.g. byexposing the polypeptide to enzymes that affect glycosylation, such asmammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

Included for use in a subject method are polypeptides that have beenmodified using ordinary chemical techniques so as to improve theirresistance to proteolytic degradation, to optimize solubilityproperties, or to render them more suitable as a therapeutic agent. Forexamples, the backbone of the peptide may be cyclized to enhancestability (see Friedler et al. (2000) J. Biol. Chem. 275:23783-23789).Analogs may be used that include residues other than naturally occurringL-amino acids, e.g. D-amino acids or non-naturally occurring syntheticamino acids. The protein may be pegylated to enhance stability. Thepolypeptides may be fused to albumin.

The polypeptides may be prepared by in vitro synthesis, usingconventional methods as known in the art, by recombinant methods, or maybe isolated from cells induced or naturally producing the protein. Theparticular sequence and the manner of preparation will be determined byconvenience, economics, purity required, and the like. If desired,various groups may be introduced into the polypeptide during synthesisor during expression, which allow for linking to other molecules or to asurface. Thus cysteines can be used to make thioethers, histidines forlinking to a metal ion complex, carboxyl groups for forming amides oresters, amino groups for forming amides, and the like.

Type II Interferon Receptor Agonists

In some embodiments, the at least one additional therapeutic agent in asubject combination therapy includes a Type II interferon receptoragonist. Thus, in some embodiments, a subject combination therapy methodcomprises administering effective amounts of an α-glucosidase inhibitorand a Type II interferon receptor agonist. As used herein, the term“Type II interferon receptor agonist” includes any naturally occurringor non-naturally-occurring ligand of a human Type II interferon receptorthat binds to and causes signal transduction via the receptor. Type IIinterferon receptor agonists include interferons, includingnaturally-occurring interferons, modified interferons, syntheticinterferons, pegylated interferons, fusion proteins comprising aninterferon and a heterologous protein, shuffled interferons; antibodyspecific for an interferon receptor; non-peptide chemical agonists; andthe like.

A specific example of a Type II interferon receptor agonist is IFN-gammaand variants thereof While the present invention exemplifies use of anIFN-gamma polypeptide, it will be readily apparent that any Type IIinterferon receptor agonist can be used in a subject method.

Interferon-Gamma

The nucleic acid sequences encoding IFN-gamma polypeptides may beaccessed from public databases, e.g., Genbank, journal publications, andthe like. While various mammalian IFN-gamma polypeptides are ofinterest, for the treatment of human disease, generally the humanprotein will be used. Human IFN-gamma coding sequence may be found inGenbank, accession numbers X13274; V00543; and NM_(—)000619. Thecorresponding genomic sequence may be found in Genbank, accessionnumbers J00219; M37265; and V00536. See, for example. Gray et al. (1982)Nature 295:501 (Genbank X13274); and Rinderknecht et al. (1984) JB.C.259:6790. In some embodiments, the IFN-γ is glycosylated.

IFN-γ1b (Actimmune®; human interferon) is a single-chain polypeptide of140 amino acids. It is made recombinantly in E. coli and isunglycosylated (Rinderknecht et al. 1984, J. Biol. Chem. 259:6790-6797).Recombinant IFN-gamma as discussed in U.S. Pat. No. 6,497,871 is alsosuitable for use herein.

The IFN-gamma to be used in a subject method may be any of naturalIFN-gamma, recombinant IFN-gamma and the derivatives thereof so far asthey have an IFN-γ activity, particularly human IFN-gamma activity.Human IFN-gamma exhibits the antiviral and anti-proliferative propertiescharacteristic of the interferons, as well as a number of otherimmunomodulatory activities, as is known in the art. Although IFN-gammais based on the sequences as provided above, the production of theprotein and proteolytic processing can result in processing variantsthereof. The unprocessed sequence provided by Gray et al., supra,consists of 166 amino acids (aa). Although the recombinant IFN-gammaproduced in E. coli was originally believed to be 146 amino acids,(commencing at amino acid 20) it was subsequently found that nativehuman IFN-gamma is cleaved after residue 23, to produce a 143 aaprotein, or 144 aa if the terminal methionine is present, as requiredfor expression in bacteria. During purification, the mature protein canadditionally be cleaved at the C terminus after reside 162 (referring tothe Gray et al. sequence), resulting in a protein of 139 amino acids, or140 amino acids if the initial methionine is present, e.g. if requiredfor bacterial expression. The N-terminal methionine is an artifactencoded by the mRNA translational “start” signal AUG that, in theparticular case of E. coli expression is not processed away. In othermicrobial systems or eukaryotic expression systems, methionine may beremoved.

For use in a subject method, any of the native IFN-gamma peptides,modifications and variants thereof, or a combination of one or morepeptides may be used. IFN-gamma peptides of interest include fragments,and can be variously truncated at the carboxyl terminus relative to thefull sequence. Such fragments continue to exhibit the characteristicproperties of human gamma interferon, so long as amino acids 24 to about149 (numbering from the residues of the unprocessed polypeptide) arepresent. Extraneous sequences can be substituted for the amino acidsequence following amino acid 155 without loss of activity. See, forexample, U.S. Pat. No. 5,690,925. Native IFN- gamma moieties includemolecules variously extending from amino acid residues 24-150; 24-151,24-152; 24- 153, 24-155; and 24-157. Any of these variants, and othervariants known in the art and having IFN-γ activity, may be used in asubject method.

The sequence of the IFN-γ polypeptide may be altered in various waysknown in the art to generate targeted changes in sequence. A variantpolypeptide will usually be substantially similar to the sequencesprovided herein, i.e., will differ by at least one amino acid, and maydiffer by at least two but not more than about ten amino acids. Thesequence changes may be substitutions, insertions or deletions. Scanningmutations that systematically introduce alanine, or other residues, maybe used to determine key amino acids. Specific amino acid substitutionsof interest include conservative and non-conservative changes.Conservative amino acid substitutions typically include substitutionswithin the following groups: (glycine, alanine); (valine, isoleucine,leucine); (aspartic acid, glutamic acid); (asparagine, glutamine);(serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine).

Modifications of interest that may or may riot alter the primary aminoacid sequence include chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation; changes in amino acid sequence thatintroduce or remove a glycosylation site; changes in amino acid sequencethat make the protein susceptible to PEGylation; and the like. IFN-gammamay be modified with one or more polyethylene glycol moieties(PEGylated). In one embodiment, the invention contemplates the use ofIFN-gamma variants with one or more non-naturally occurringglycosylation and/or pegylation sites that are engineered to provideglycosyl- and/or PEG-derivatized polypeptides with reduced serumclearance, such as the IFN-gamma polypeptide variants described inInternational Patent Publication No. WO 01/36001 and WO 02/081507. Alsoincluded are modifications of glycosylation, e.g., those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps; e.g., byexposing the polypeptide to enzymes that affect glycosylation, such asmammalian glycosylating or deglycosylatirig enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.,phosphotyrosine, phosphoserine, or phosphothreonine.

Included for use in the subject invention are IFN-γ polypeptides thathave been modified using ordinary chemical techniques so as to improvetheir resistance to proteolytic degradation, to optimize solubilityproperties, or to render them more suitable as a therapeutic agent. Forexamples, the backbone of the peptide may be cyclized to enhancestability (see, for example, Friedler et al. 2000, J. Biol. Chem.275:23783-23789). Analogs may be used that include residues other thannaturally occurring L-amino acids, e.g., D-amino acids or non-naturallyoccurring synthetic amino acids. The protein may be pegylated to enhancestability.

In some embodiments, the present invention contemplates use of a knownhyperglycosylated polypeptide variant of a parent protein therapeutic.In some embodiments, the parent protein therapeutic is an interferon,and a known hyperglycosylated polypeptide variant comprises (1) acarbohydrate moiety covalently attached to at least one non-nativeglycosylation site not found in the parent interferon and/or (2) acarbohydrate moiety covalently attached to at least one nativeglycosylation site found but not glycosylated in the parent interferon.

In some embodiments, the parent polypeptide is a Type II interferonreceptor polypeptide agonist. Type II interferon receptor polypeptideagonists include interferon-gamma (IFN-γ). Thus, e.g., a knownhyperglycosylated polypeptide variant can be a hyperglycosylated Type IIinterferon receptor polypeptide agonist variant, includinghyperglycosylated IFN-γ.

Any of the native IFN-gamma peptides, modifications and variantsthereof, or a combination of one or more peptides can serve as a parentpolypeptide referent in connection with the present methods and/orcompositions. IFN-gamma peptides of interest include fragments, and canbe variously truncated at the carboxyl terminus relative to the fullsequence. Such fragments continue to exhibit the characteristicproperties of human gamma interferon, so long as amino acids 24 to about149 (numbering from the residues of the unprocessed polypeptide) arepresent. Extraneous sequences can be substituted for the amino acidsequence following amino acid 155 without loss of activity. See, forexample, U.S. Pat. No. 5,690,925. Native IFN-gamma moieties includemolecules variously extending from amino acid residues 24-150; 24-151,24-152; 24- 153, 24-155; and 24-157.

Any known hyperglycosylated IFN-gamma polypeptide variant that retains adesired pharmacologic activity of a parent IFN-gamma polypeptide may beused in the methods and/or compositions of the invention.

In one aspect, the parent polypeptide is the mature, native IFN-gammapolypeptide; and the hyperglycosylated polypeptide variant of the parentpolypeptide is an [S99T]IFN-gamma glycopeptide, where the[S99T]IFN-gamma glycopeptide is a variant of the mature, nativeIFN-gamma having (a) a threonine residue substituted for the nativeserine residue at amino acid position 99 in the amino acid sequence ofIFN-gamma and (b) a carbohydrate moiety covalently attached to theR-group of the asparagine residue at amino acid position 97 in the aminoacid sequence of (a).

Since the glycosylation site formed by N97, Y98, T99 in the[S99T]IFN-gamma variant is different than the glycosylation site formedby N97, Y98, S99 in native IFN-gamma, the N97, Y98, T99 glycosylationsite qualifies as a non-native glycosylation site not found in theparent polypeptide. In addition, as described in WO 02/081507, the S99Tsubstitution in the amino acid sequence of native IFN-gamma provides forgreater efficiency of glycosylation at the N97, Y98, T99 glycosylationsite in the [S99T]IFN-gamma variant compared to the efficiency ofglycosylation at the N97, Y98, S99 glycosylation site in nativeIFN-gamma. Thus, [S99T]IFN-gamma qualifies as a hyperglycosylatedpolypeptide variant of the parent IFN-gamma polypeptide.

In another aspect, the parent polypeptide is mature, native IFN-gamma;and the hyperglycosylated polypeptide variant of the parent polypeptideis an [E38N]IFN-gamma glycopeptide, where the [E38N]IFN-gammaglycopeptide is a variant of the mature, native IFN-gamma having (a) anasparagine residue substituted for the native glutamic acid residue atamino acid position 38 in the amino acid sequence of IFN-gamma and (b) acarbohydrate moiety covalently attached to the R-group of the asparagineresidue at amino acid position 38 in the amino acid sequence of (a).

In another aspect, the parent polypeptide is mature, native IFN-gamma;and the hyperglycosylated polypeptide variant of the parent polypeptideis an [E38N, S99T]IFN-gamma glycopeptide, where the [E38N,S99T]IFN-gamma glycopeptide is a variant of the mature, native IFN-gammahaving (a) asparagine and threonine residues substituted for the nativeglutamic acid and serine residues at amino acid positions 38 and 99 inthe amino acid sequence of IFN-gamma and (b) a carbohydrate moietycovalently attached to the R-group of the asparagine residue at each ofamino acid positions 38 and 97 in the amino acid sequence of (a).

In another aspect, the parent polypeptide is mature, native IFN-gamma;and the hyperglycosylated polypeptide variant of the parent polypeptideis an [E38N, S40T]IFN-gamma glycopeptide, where the [E38N,S40T]IFN-gamma glycopeptide is a variant of the mature, native IFN-gammahaving (a) asparagine and threonine residues substituted for the nativeglutamic acid and serine residues at amino acid positions 38 and 40 inthe amino acid sequence of IFN-gamma and (b) a carbohydrate moietycovalently attached to the R-group of the asparagine residue at aminoacid position 38 in the amino acid sequence (a).

In another aspect, the parent polypeptide is mature, native IFN-gammahaving the amino acid sequence; and the hyperglycosylated polypeptidevariant of the parent polypeptide is an [E38N, S40T, S99T]IFN-gammaglycopeptide, where the [E38N, S40T, S99T]IFN-gamma glycopeptide i s avariant of the mature, native IFN-gamma having (a) asparagine, threonineand threonine residues substituted for the native glutamic acid, serineand serine residues at amino acid positions 38, 40 and 99, respectively,in the amino acid sequence of IFN-gamma and (b) a carbohydrate moietycovalently attached to the R-group of the asparagine residue at aminoacid position 38 in the amino acid sequence of (a), and optionallyfurther having (c) a carbohydrate moiety covalently attached to theR-group of the asparagine residue at amino acid position 97 in the aminoacid sequence of IFN-gamma.

In another aspect, a known hyperglycosylated polypeptide variant of aparent interferon-gamma therapeutic differs from the parentinterferon-gamma therapeutic to the extent that the knownhyperglycosylated polypeptide variant comprises (1) a carbohydratemoiety covalently attached to a non-native glycosylation site not foundin the parent interferon-gamma therapeutic and/or (2) a carbohydratemoiety covalently attached to a native glycosylation site found but notglycosylated in the parent interferon-gamma therapeutic.

In another aspect, the parent protein therapeutic is interferon gamma-1band the known hyperglycosylated polypeptide variant of the parentinterferon gamma-1b therapeutic is glycosylated native (wild-type) humanIFN-γ as described in WO 02/081507.

The IFN-γ polypeptides may be prepared by in vitro synthesis, usingconventional methods as known in the art, by recombinant methods, or maybe isolated from cells induced or naturally producing the protein. Theparticular sequence and the manner of preparation will be determined byconvenience, economics, purity required, and the like. If desired,various groups may be introduced into the polypeptide during synthesisor during expression, which allow for linking to other molecules or to asurface. Thus cysteines can be used to make thioethers, histidines forlinking to a metal ion complex, carboxyl groups for forming amides oresters, amino groups for forming amides, and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using a liquidchromatography method (e.g., high performance liquid chromatography),size exclusion chromatography, gel electrophoresis, affinitychromatography, or other purification technique. For the most part, thecompositions which are used will comprise at least 20% by weight of thedesired product, more usually at least about 75% by weight, preferablyat least about 95% by weight, and for therapeutic purposes, usually atleast about 99.5% by weight, in relation to contaminants related to themethod of preparation of the product and its purification. Usually, thepercentages will be based upon total protein.

Ribavirin

In some embodiments, the at least one additional suitable therapeuticagent includes ribavirin. Ribavirin,1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available from ICNPharmaceuticals, Inc., Costa Mesa, Calif., is described in the MerckIndex, compound No. 8199, Eleventh Edition. Its manufacture andformulation is described in U.S. Pat. No. 4,211,771. The invention alsocontemplates use of derivatives of ribavirin (see, e.g., U.S. Pat. No.6,277,830). The ribavirin may be administered orally in capsule ortablet form, or in the same or different administration form and in thesame or different route as the α-glucosidase inhibitor. Of course, othertypes of administration of both medicaments, as they become availableare contemplated, such as by nasal spray, transdermally, by suppository,by sustained release dosage form, etc. Any form of administration willwork so long as the proper dosages are delivered without destroying theactive ingredient.

Ribavirin is generally administered in an amount ranging from about 400mg to about 1200 mg, from about 600 mg to about 1000 mg, or from about700 to about 900 mg per day. In some embodiments, ribavirin isadministered throughout the entire course of α-glucosidase inhibitortherapy. In other embodiments, ribavirin is administered only during thefirst period of time. In still other embodiments, ribavirin isadministered only during the second period of time.

Levovirin

In some embodiments, the at least one additional suitable therapeuticagent includes levovirin. Levovirin is the L-enantiomer of ribavirin,and exhibits the property of enhancing a Th1 immune response over a Th2immune response. Levovirin is manufactured by ICN Pharmaceuticals.

Levovirin has the following structure:

Viramidine

In some embodiments, the at least one additional suitable therapeuticagent includes viramidine. Viramidine is a 3-carboxamidine derivative ofribavirin, and acts as a prodrug of ribavirin. It is efficientlyconverted to ribavirin by adenosine deaminases.

Viramidine has the following structure:

Nucleoside Analogs

Nucleoside analogs that are suitable for use in a subject combinationtherapy include, but are not limited to, ribavirin, levovirin,viramidine, isatoribine, an L-ribofuranosyl nucleoside as disclosed inU.S. Pat. No. 5,559,101 and encompassed by Formula I of U.S. Pat. No.5,559,101 (e.g., 1-β-L-ribofuranosyluracil,1-β-L-ribofuranosyl-5-fluorouracil, 1-β-L-ribofuranosylcytosine,9-β-L-ribofuranosyladenine, 9-β-L-ribofuranosylhypoxanthine,9-β-L-ribofuraosylguanine, 9-β-L-ribofuranosy1-6-thioguanine,2-amino-α-L-ribofuran[1′,2′:4,5]oxazoline,O²,O²-anhydro-1-α-L-ribofuranosyluracil, 1-α-L-ribofuranosyluracil,1-(2,3,5-tri-O-benzoyl-α-ribofuranosyl)-4-thiouracil,1-α-L-ribofuranosylcytosine, 1-α-L-ribofuranosyl-4-thiouracil,1-α-L-ribofuranosyl-5-fluorouracil,2-amino-β-L-arabinofurano[1′,2′:4,5]oxazoline,O²,O²-anhydro-β-L-arabinofuranosyluracil, 2′-deoxy-β-L-uridine,3′5′-Di-O-benzoyl-2′deoxy-4-thio β-L-uridine, 2′-deoxy-β-L-cytidine,2′-deoxy-β-L-4-thiouridine, 2′-deoxy-β-L-thymidine,2′-deoxy-β-L-5-fluorouridine, 2′,3′-dideoxy-β-L-uridine,2′-deoxy-β-L-5-fluorouridine, and 2′-deoxy-β-L-inosine); a compound asdisclosed in U.S. Pat. No. 6,423,695 and encompassed by Formula I ofU.S. Pat. No. 6,423,695; a compound as disclosed in U.S. PatentPublication No. 2002/0058635, and encompassed by Formula 1 of U.S.Patent Publication No. 2002/0058635; a nucleoside analog as disclosed inWO 01/90121 A2 (Idenix); a nucleoside analog as disclosed in WO02/069903 A2 (Biocryst Pharmaceuticals Inc.); a nucleoside analog asdisclosed in WO 02/057287 A2 or WO 02/057425 A2 (both Merck/Isis); andthe like.

HCV NS3 inhibitors

In some embodiments, the at least one additional suitable therapeuticagent includes HCV NS3 inhibitors. Suitable HCV non-structural protein-3(NS3) inhibitors include, but are not limited to, a tri-peptide asdisclosed in U.S. Pat. Nos. 6,642,204, 6,534,523, 6,420,380, 6,410,531,6,329,417, 6,329,379, and 6,323,180 (Boehringer-Ingelheim); a compoundas disclosed in U.S. Pat. No. 6,143,715 (Boehringer-Ingelheim); amacrocyclic compound as disclosed in U.S. Pat. No. 6,608,027(Boehringer-Ingelheim); an NS3 inhibitor as disclosed in U.S. Pat. Nos.6,617,309, 6,608,067, and 6,265,380 (Vertex Pharmaceuticals); anazapeptide compound as disclosed in U.S. Pat. No. 6,624,290 (Schering);a compound as disclosed in U.S. Pat. No. 5,990,276 (Schering); acompound as disclosed in Pause et al. (2003) J. Biol. Chem.278:20374-20380; NS3 inhibitor BILN 2061 (Boehringer-Ingelheim; Lamarreet al. (2002) Hepatology 36:301A; and Lamarre et al. (Oct. 26, 2003)Nature doi:10.1038/nature02099); NS3 inhibitor VX-950 (VertexPharmaceuticals; Kwong et al. (Oct. 24-28, 2003) 54^(th) Ann. MeetingAASLD); NS3 inhibitor SCH6 (Abib et al. (Oct. 24-28, 2003) Abstract 137.Program and Abstracts of the 54^(th) Annual Meeting of the AmericanAssociation for the Study of Liver Diseases (AASLD). Oct. 24-28, 2003.Boston, Mass.); any of the NS3 protease inhibitors disclosed in WO99/07733, WO 99/07734, WO 00/09558, WO 00/09543, WO 00/59929 or WO02/060926 (e.g., compounds 2, 3, 5, 6, 8, 10, 11, 18, 19, 29, 30, 31,32, 33, 37, 38, 55, 59, 71, 91, 103, 104, 105, 112, 113, 114, 115, 116,120, 122, 123, 124, 125, 126 and 127 disclosed in the table of pages224-226 in WO 02/060926); an NS3 protease inhibitor as disclosed in anyone of U.S. Patent Publication Nos. 2003019067, 20030187018, and20030186895; and the like.

Of particular interest in many embodiments are NS3 inhibitors that arespecific NS3 inhibitors, e.g., NS3 inhibitors that inhibit NS3 serineprotease activity and that do not show significant inhibitory activityagainst other serine proteases such as human leukocyte elastase, porcinepancreatic elastase, or bovine pancreatic chymotrypsin, or cysteineproteases such as human liver cathepsin B.

NS5B Inhibitors

In some embodiments, the at least one additional suitable therapeuticagent includes NS5B inhibitors. Suitable HCV non-structural protein-5(NS5; RNA-dependent RNA polymerase) inhibitors include, but are notlimited to, a compound as disclosed in U.S. Pat. No. 6,479,508(Boehringer-Ingelheim); a compound as disclosed in any of InternationalPatent Application Nos. PCT/CA02/01127, PCT/CA02/01128, andPCT/CA02/01129, all filed on Jul. 18, 2002 by Boehringer Ingelheim; acompound as disclosed in U.S. Pat. No. 6,440,985 (ViroPharma); acompound as disclosed in WO 01/47883, e.g., JTK-003 (Japan Tobacco); adinucleotide analog as disclosed in Zhong et al. (2003) Antimicrob.Agents Chemother. 47:2674-2681; a benzothiadiazine compound as disclosedin Dhanak et al. (2002) J. Biol Chem. 277(41):38322-7; an NS5B inhibitoras disclosed in WO 02/100846 A1 or WO 02/100851 A2 (both Shire); an NS5Binhibitor as disclosed in WO 01/85172 A1 or WO 02/098424 Al (both GlaxoSmithKline); an NS5B inhibitor as disclosed in WO 00/06529 or WO02/06246 A1 (both Merck); an NS5B inhibitor as disclosed in WO 03/000254(Japan Tobacco); an NS5B inhibitor as disclosed in EP 1 256,628 A2(Agouron); JTK-002 (Japan Tobacco); JTK-109 (Japan Tobacco); and thelike.

Of particular interest in many embodiments are NS5 inhibitors that arespecific

NS5 inhibitors, e.g., NS5 inhibitors that inhibit NS5 RNA-dependent RNApolymerase and that lack significant inhibitory effects toward other RNAdependent RNA polymerases and toward DNA dependent RNA polymerases.

Additional Antiviral Therapeutic Agents

Additional antiviral therapeutic agents that can be administered in asubject combination therapy include, but are not limited to, inhibitorsof inosine monophosphate dehydrogenase (IMPDH); ribozymes that arecomplementary to viral nucleotide sequences; antisense RNA inhibitors;and the like.

IMPDH Inhibitors

IMPDH inhibitors that are suitable for use in a subject combinationtherapy include, but are not limited to, VX-497((S)-N-3-[3-(3-methoxy-4-oxazol-5-yl-phenyl)-ureido]-benzyl-carbamicacid tetrahydrofuran-3-yl-ester); Vertex Pharmaceuticals; see, e.g.,Markland et al. (2000) Antimicrob. Agents Chemother. 44:859-866);ribavirin; levovirin (Ribapharm; see, e.g., Watson (2002) Curr OpinInvestig Drugs 3(5):680-3); viramidine (Ribapharm); and the like.

Ribozyme and Antisense

Ribozyme and antisense antiviral agents that are suitable for use in asubject combination therapy include, but are not limited to, ISIS 14803(ISIS Pharmaceuticals/Elan Corporation; see, e.g., Witherell (2001) CurrOpin Investig Drugs. 2(11):1523-9); Heptazyme™; and the like.

Side Effect Management Agents

In some embodiments, a subject therapy further comprises administering apalliative agent (e.g., an agent that reduces patient discomfort causedby a therapeutic agent), or other agent for the avoidance, treatment, orreduction of a side effect of a therapeutic agent. Such agents are alsoreferred to as “side effect management agents.” Suitable side effectmanagement agents include agents for the avoidance, treatment, orreduction of a side effect of an agent that inhibits enzymatic activityof a membrane-bound α-glucosidase; agents for the avoidance, treatment,or reduction of a side effect of a Type I interferon receptor agonist;agents for the avoidance, treatment, or reduction of a side effect of aType II interferon receptor agonist; and the like.

Suitable side effect management agents include agents that are effectivein pain management; agents that ameliorate gastrointestinal discomfort;analgesics, anti-inflammatories, antipsychotics, antineurotics,anxiolytics, and hematopoietic agents. In addition, the inventioncontemplates the use of any compound for palliative care of patientssuffering from pain or any other side effect in the course of treatmentwith a subject therapy. Exemplary palliative agents includeacetaminophen, ibuprofen, and other NSAIDs, H2 blockers, and antacids.

Analgesics that can be used to alleviate pain in the methods of theinvention include non-narcotic analgesics such as non-steroidalanti-inflammatory drugs (NSAIDs) acetaminophen, salicylate,acetyl-salicylic acid (aspirin, diflunisal), ibuprofen, Motrin,Naprosyn, Nalfon, and Trilisate, indomethacin, glucametacine,acemetacin, sulindac, naproxen, piroxicam, diclofenac, benoxaprofen,ketoprofen, oxaprozin, etodolac, ketorolac tromethamine, ketorolac,nabumetone, and the like, and mixtures of two or more of the foregoing.

Other suitable analgesics include fentanyl, buprenorphine, codeinesulfate, morphine hydrochloride, codeine, hydromorphone (Dilaudid),levorphanol (Levo-Dromoran), methadone (Dolophine), morphine, oxycodone(in Percodan), and oxymorphone (Numorphan). Also suitable for use arebenzodiazepines including, but not limited to, flurazepam (Dalmane),diazepam (Valium), and Versed, and the like.

Anti-Inflammatory Agents

Suitable anti-inflammatory agents include, but are not limited to,steroidal anti-inflammatory agents, and non-steroidal anti-inflammatoryagents.

Suitable steroidal anti-inflammatory agents include, but are not limitedto, hydrocortisone, hydroxyltriamcinolone., alpha-methyl dexamethasone,dexamethasone-phosphate, beclomethasone dipropionate, clobetasolvalerate, desonide, desoxymethasone, desoxycorticosterone acetate,dexamethasone, dichlorisone, diflorasone diacetate, diflucortolonevalerate, fluadrenolone, fluclorolone acetonide, fludrocortisone,flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortinebutylester, fluocortolone, fluprednidene (fluprednylidene) acetate,flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisonebutyrate, methylprednisolone, triamcinolone acetonide, conisone,cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,fluradrenolone acetonide, medrysone, amcinafel, amcinafide,betamethasone and the balance of its esters, chloroprednisone,chlorprednisone acetate, clocortelone, clescinolone, dichlorisone,difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone,fluprednisolone, hydrocortisone valerate, hydrocortisonecyclopentylpropionate, hydrocortamate, meprednisone, paramethasone,prednisolone, prednisone, beclomethasone dipropionate, triamcinolone,and mixtures of two or more of the foregoing.

Suitable non-steroidal anti-inflammatory agents, include, but are notlimited to, 1) the oxicams, such as piroxicam, isoxicam, tenoxicam, andsudoxicam; 2) the salicylates, such as aspirin, disalcid, benorylate,trilisate, safapryn, solprin, and fendosal; 3) the acetic acidderivatives, such as diclofenac, fenclofenac, indomethacin, sulindac,tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin,fentiazac, zomepiract, clidanac, oxepinac, and felbinac; 4) thefenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, andtolfenamic acids; 5) the propionic acid derivatives, such as ibuprofen,naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen,indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen,tioxaprofen, suprofen, al:minoprofen, and tiaprofenic; and 6) thepyrazoles, such as phenylbutazone, oxyphenbutazone, feprazone,azapropazone, and trimethazone, mixtures of these non-steroidalanti-inflammatory agents may also be employed, as well as thepharmaceutically-acceptable salts and esters of these agents.

Suitable anti-inflammatory agents include, but are not limited to,Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; AlphaAmylase; Amcinafal; Amcinafide; Amfenac Sodium; AmipriloseHydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; BalsalazideDisodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains;Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen;Clobetasol Propionate; Clobetasone Butyrate; Clopirac; CloticasonePropionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide;Desoximetasone; -Dexamethasone Dipropionate; Diclofenac Potassium;Diclofenac Sodium; Diflorasone Diacetate; -Diflumidone Sodium;Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide;Endrysone; Enlimomab Enolicam Sodium; Epirizole; Etodolac; Etofenamate;Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal;Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid;Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; FluocortinButyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen;Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; HalobetasolPropionate; Halopredone Acetate; Ibufenac; Ibuprofen; IbuprofenAluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; IndomethacinSodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate;Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam;Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid;Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone;Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen;Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein;Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride;Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Piroxicam;Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate;Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate;Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; SanguinariumChloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen;Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; TenidapSodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; TixocortolPivalate; Tolmetin; Tolmetin. Sodium; Triclonide; Triflumidate;Zidometacin; Zomepirac Sodium.

Antipsychotic and antineurotic drugs that can be used to alleviatepsychiatric side effects in the methods of the invention include any andall selective serotonin receptor inhibitors (SSRIs) and otheranti-depressants, anxiolytics (e.g. alprazolam), etc. Anti-depressantsinclude, but are not limited to, serotonin reuptake inhibitors such asCelexa®, Desyrel®, Effexor®, Luvox®, Prozac®, Zoloft®, and Serzone®;tricyclics such as Adapin®, Anafrinil®, Elavil®, Janimmine®, Ludiomil®,Pamelor®, Tofranil®, Vivactil®, Sinequan®, and Surmontil®; monoamineoxidase inhibitors such as Eldepryl®, Marplan®, Nardil®, and Parnate®.Anti-anxiety agents include, but are not limited to, azaspirones such asBuSpar®, benzodiazepines such as Ativan®, Librium®, Tranxene®, Centrax®,Klonopin®, Paxipam®, Serax®, Valium®, and Xanax®; and beta-blockers suchas Inderal® and Tenormin®.

Agents that reduce gastrointestinal discomfort such as nausea, diarrhea,gastrointestinal cramping, and the like are suitable palliative agentsfor use in a subject combination therapy. Suitable agents include, butare not limited to, antiemetics, anti-diarrheal agents, H2 blockers,antacids, and the like.

Suitable H2 blockers (histamine type 2 receptor antagonists) that aresuitable for use as a palliative agent in a subject therapy include, butare not limited to, Cimetidine (e.g., Tagamet, Peptol, Nu-cimet,apo-cimetidine, non-cimetidine); Ranitidine (e.g., Zantac, Nu-ranit,Novo-randine, and apo-ranitidine); and Famotidine (Pepcid,Apo-Famotidine, and Novo-Famotidine).

Suitable antacids include, but are not limited to, aluminum andmagnesium hydroxide (Maalox®, Mylanta®); aluminum carbonate gel(Basajel®); aluminum hydroxide (Amphojel®, AlternaGEL®); calciumcarbonate (Tums®, Titralac®); magnesium hydroxide; and sodiumbicarbonate.

Antiemetics include, but are not limited to, 5-hydroxytryptophan-3(5HT3) inhibitors; corticosteroids such as dexamethasone andmethylprednisolone; Marinol® (dronabinol); prochlorperazine;benzodiazepines; promethazine; and metoclopramide cisapride; AlosetronHydrochloride; Batanopride Hydrochloride; Bemesetron; Benzquinamide;Chlorpromazine; Chlorpromazine Hydrochloride; Clebopride; CyclizineHydrochloride; Dimenhydrinate; Diphenidol; Diphenidol Hydrochloride;Diphenidol Pamoate; Dolasetron Mesylate; Domperidone; Dronabinol;Fludorex; Flumeridone; Galdansetron Hydrochloride; Granisetron;Granisetron Hydrochloride; Lurosetron Mesylate; Meclizine Hydrochloride;Metoclopramide Hydrochloride; Metopimazine; Ondansetron Hydrochloride;Pancopride; Prochlorperazine; Prochlorperazine Edisylate;Prochlorperazine Maleate; Promethazine Hydrochloride; Thiethylperazine;Thiethylperazine Malate; Thiethylperazine Maleate; TrimethobenzamideHydrochloride; Zacopride Hydrochloride.

Anti-diarrheal agents include, but are not limited to, Rolgamidine,Diphenoxylate hydrochloride (Lomotil), Metronidazole (Flagyl),Methylprednisolone (Medrol), Sulfasalazine (Azulfidine), and the like.

Suitable hematopoietic agents that can be used to prevent or restoredepressed blood cell populations in the methods of the invention includeerythropoietins, such as EPOGEN™ epoetin-alfa, granulocyte colonystimulating factors (G-CSFs), such as NEUPOGEN™ filgrastim,granulocyte-macrophage colony stimulating factors (GM-CSFs),thrombopoietins, etc.

Dosages, Formulations, and Routes of Administration

An active agent (e.g., an α-glucosidase inhibitor, at least oneadditional therapeutic agent, etc.) is administered to individuals in aformulation with a pharmaceutically acceptable excipient(s). The terms“active agent” and “therapeutic agent” are used interchangeably herein.A wide variety of pharmaceutically acceptable excipients are known inthe art and need not be discussed in detail herein. Pharmaceuticallyacceptable excipients have been amply described in a variety ofpublications, including, for example, A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy,” 20^(th) edition, Lippincott,Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug DeliverySystems (1999) H.C. Ansel et al., eds., 7^(th) ed., Lippincott,Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3^(rd) ed. Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

In the subject methods, an active agent (e.g., an α-glucosidaseinhibitor, at least one additional therapeutic agent, etc.) may beadministered to the host using any convenient means capable of resultingin the desired therapeutic effect. Thus, an active agent can beincorporated into a variety of formulations for therapeuticadministration. More particularly, an active agent can be formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols.

As such, administration of an active agent can be achieved in variousways, including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, subcutaneous, intramuscular, transdermal, intratracheal,etc., administration. In some embodiments, two different routes ofadministration are used. For example, in some embodiments, anα-glucosidase inhibitor is administered orally, while IFN-γ or IFN-α isadministered subcutaneously.

Subcutaneous administration of an active agent (e.g., an α-glucosidaseinhibitor, at least one additional therapeutic agent, etc.) can beaccomplished using standard methods and devices, e.g., needle andsyringe, a subcutaneous injection port delivery system, and the like.See, e.g., U.S. Pat. Nos. 3,547,119; 4,755,173; 4,531,937; 4,311,137;and 6,017,328. A combination of a subcutaneous injection port and adevice for administration of a therapeutic agent to a patient throughthe port is referred to herein as “a subcutaneous injection portdelivery system.” In some embodiments, subcutaneous administration isachieved by a combination of devices, e.g., bolus delivery by needle andsyringe, followed by delivery using a continuous delivery system.

In some embodiments, an active agent (e.g., an α-glucosidase inhibitor,at least one additional therapeutic agent, etc.) is delivered by acontinuous delivery system. The terms “continuous delivery system,”“controlled delivery system,” and “controlled drug delivery device,” areused interchangeably to refer to controlled drug delivery devices, andencompass pumps in combination with catheters, injection devices, andthe like, a wide variety of which are known in the art.

Mechanical or electromechanical infusion pumps can also be suitable foruse with the present invention. Examples of such devices include thosedescribed in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019;4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; andthe like. In general, the present methods of drug delivery can beaccomplished using any of a variety of refillable, pump systems. Pumpsprovide consistent, controlled release over time. Typically, the agentis in a liquid formulation in a drug-impermeable reservoir, and isdelivered in a continuous fashion to the individual.

In one embodiment, the drug delivery system is an at least partiallyimplantable device. The implantable device can be implanted at anysuitable implantation site using methods and devices well known in theart. An implantation site is a site within the body of a subject atwhich a drug delivery device is introduced and positioned. Implantationsites include, but are not necessarily limited to a subdermal,subcutaneous, intramuscular, or other suitable site within a subject'sbody. Subcutaneous implantation sites are generally used because ofconvenience in implantation and removal of the drug delivery device.

Drug release devices suitable for use in the invention may be based onany of a variety of modes of operation. For example, the drug releasedevice can be based upon a diffusive system, a convective system, or anerodible system (e.g., an erosion-based system). For example, the drugrelease device can be an electrochemical pump, osmotic pump, anelectroosmotic pump, a vapor pressure pump, or osmotic bursting matrix,e.g., where the drug is incorporated into a polymer and the polymerprovides for release of drug formulation concomitant with degradation ofa drug-impregnated polymeric material (e.g., a biodegradable,drug-impregnated polymeric material). In other embodiments, the drugrelease device is based upon an electrodiffusion system, an electrolyticpump, an effervescent pump, a piezoelectric pump, a hydrolytic system,etc.

Drug release devices based upon a mechanical or electromechanicalinfusion pump are also suitable for use with the present invention.Examples of such devices include those described in, for example, U.S.Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and thelike. In general, a subject treatment method can be carried out usingany of a variety of refillable, non-exchangeable pump systems. Pumps andother convective systems are generally preferred due to their generallymore consistent, controlled release over time. Osmotic pumps are used insome embodiments due to their combined advantages of more consistentcontrolled release and relatively small size (see, e.g., PCT publishedapplication no. WO 97/27840 and U.S. Pat. Nos. 5,985,305 and5,728,396)). Exemplary osmotically-driven devices suitable for use in asubject treatment method include, but are not necessarily limited to,those described in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899;3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228;4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725;4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727;5,234,692; 5,234,693; 5,728,396; and the like.

In some embodiments, the drug delivery device is an implantable device.The drug delivery device can be implanted at any suitable implantationsite using methods and devices well known in the art. As noted above, animplantation site is a site within the body of a subject at which a drugdelivery device is introduced and positioned. Implantation sitesinclude, but are not necessarily limited to a subdermal, subcutaneous,intramuscular, or other suitable site within a subject's body.

In some embodiments, a therapeutic agent is delivered using animplantable drug delivery system, e.g., a system that is programmable toprovide for administration of a therapeutic agent. Exemplaryprogrammable, implantable systems include implantable infusion pumps.Exemplary implantable infusion pumps, or devices useful in connectionwith such pumps, are described in, for example, U.S. Pat. Nos.4,350,155; 5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276;6,241,704; 6,464,687; 6,475,180; and 6,512,954. A further exemplarydevice that can be adapted for the present invention is the Synchromedinfusion pump (Medtronic).

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

For oral preparations, an active agent (e.g., an α-glucosidaseinhibitor, at least one additional therapeutic agent, etc.) isformulated alone or in combination with appropriate additives to maketablets, powders, granules or capsules, for example, with conventionaladditives, such as lactose, mannitol, corn starch or potato starch; withbinders, such as crystalline cellulose, cellulose derivatives, acacia,corn starch or gelatins; with disintegrators, such as corn starch,potato starch or sodium carboxymethylcellulose; with lubricants, such astalc or magnesium stearate; and if desired, with diluents, bufferingagents, moistening agents, preservatives, and flavoring agents.

Furthermore, an active agent can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. An active agent can be administered rectally via a suppository.The suppository can include vehicles such as cocoa butter, carbowaxesand polyethylene glycols, which melt at body temperature, yet aresolidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more activeagents. Similarly, unit dosage forms for injection or intravenousadministration may comprise the agent(s) in a composition as a solutionin sterile water, normal saline or another pharmaceutically acceptablecarrier.

Dosages

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of an activeagent (e.g., an α-glucosidase inhibitor, at least one additionaltherapeutic agent, etc.) calculated in an amount sufficient to producethe desired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms depend on the particular agent employed and the effect to beachieved, and the pharmacodynamics associated with each agent in thehost.

Monotherapies

An agent that inhibits enzymatic activity of a membrane-boundα-glucosidase is useful in the treatment of a flavivirus infection,e.g., HCV infection, WNV infection, etc; as well as in the treatment ofliver fibrosis that may occur as a result of, e.g., an HCV infection. Asubject method that provides for administration of an agent thatinhibits enzymatic activity of a membrane-bound α-glucosidase is alsoreferred to herein as “an α-glucosidase inhibitor treatment” or “theα-glucosidase inhibitor treatment.”

In many embodiments, an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase is administered for a period of about 1 dayto about 7 days, or about 1 week to about 2 weeks, or about 2 weeks toabout 3 weeks, or about 3 weeks to about 4 weeks, or about 1 month toabout 2 months, or about 3 months to about 4 months, or about 4 monthsto about 6 months, or about 6 months to about 8 months, or about 8months to about 12 months, or at least one year, and may be administeredover longer periods of time.

An agent that inhibits enzymatic activity of a membrane-boundα-glucosidase can be administered 5 times per day, 4 times per day, tid(three times daily), bid, qd, qod, biw, tiw, qw, qow, three times permonth, or once monthly. In other embodiments, an agent that inhibitsenzymatic activity of a membrane-bound α-glucosidase is administered asa continuous infusion.

In many embodiments, an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase is administered orally.

In connection with the above-described methods for the treatment of aflavivirus infection, treatment of HCV infection, treatment of WNVinfection, and treatment of liver fibrosis that occurs as a result of anHCV infection, an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase is administered to the patient at a dosageof from about 30 mg per day to about 600 mg per day in divided doses,e.g., from about 30 mg per day to about 60 mg per day, from about 60 mgper day to about 75 mg per day, from about 75 mg per day to about 90 mgper day, from about 90 mg per day to about 120 mg per day, from about120 mg per day to about 150 mg per day, from about 150 mg per day toabout 180 mg per day, from about 180 mg per day to about 210 mg per day,from about 210 mg per day to about 240 mg per day, from about 240 mg perday to about 270 mg per day, from about 270 mg per day to about 300 mgper day, from about 300 mg per day to about 360 mg per day, from about360 mg per day to about 420 mg per day, from about 420 mg per day toabout 480 mg per day, or from about 480 mg to about 600 mg per day.

In some embodiments, an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase is administered in a dosage of about 25 mgthree times daily. In some embodiments, an agent that inhibits enzymaticactivity of a membrane-bound α-glucosidase is administered in a dosageof about 50 mg three times daily. In some embodiments, an agent thatinhibits enzymatic activity of a membrane-bound α-glucosidase isadministered in a dosage of about 100 mg three times daily. In someembodiments, an agent that inhibits enzymatic activity of amembrane-bound α-glucosidase is administered in a dosage of about 75 mgper day to about 150 mg per day in two or three divided doses, where theindividual weighs 60 kg or less. In some embodiments, an agent thatinhibits enzymatic activity of a membrane-bound α-glucosidase isadministered in a dosage of about 75 mg per day to about 300 mg per dayin two or three divided doses, where the individual weighs 60 kg ormore.

The amount of active ingredient (e.g., an agent that inhibits enzymaticactivity of a membrane-bound α-glucosidase) that may be combined withcarrier materials to produce a dosage form can vary depending on thehost to be treated and the particular mode of administration. A typicalpharmaceutical preparation can contain from about 5% to about 95% activeingredient (w/w), and in some cases from about 95% to about 98%, or fromabout 98% to about 99% (w/w) active ingredient). In other embodiments,the pharmaceutical preparation can contain from about 20% to about 80%active ingredient.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific agent that inhibits enzymatic activity of amembrane-bound α-glucosidase, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven agent that inhibits enzymatic activity of a membrane-boundα-glucosidase are readily determinable by those of skill in the art by avariety of means. A typical means is to measure the physiologicalpotency of a given active agent.

In many embodiments, multiple doses of an agent that inhibits enzymaticactivity of a membrane-bound α-glucosidase are administered. Forexample, an agent that inhibits enzymatic activity of a membrane-boundα-glucosidase is administered once per month, twice per month, threetimes per month, every other week (qow), once per week (qw), twice perweek (biw), three times per week (tiw), four times per week, five timesper week, six times per week, every other day (qod), daily (qd), twice aday (qid), or three times a day (tid), over a period of time rangingfrom about one day to about one week, from about two weeks to about fourweeks, from about one month to about two months, from about two monthsto about four months, from about four months to about six months, fromabout six months to about eight months, from about eight months to about1 year, from about 1 year to about 2 years, or from about 2 years toabout 4 years, or more.

Methods of Treating a Flavivirus Infection

The present invention provides methods of treating a flavivirusinfection by administering to an individual in need thereof atherapeutically effective amount of an agent that inhibits enzymaticactivity of a membrane-bound α-glucosidase. Individuals who are to betreated according to the methods of the invention include individualswho have been clinically diagnosed with a flavivirus infection, as wellas individuals who exhibit one or more of the signs and the symptoms ofclinical infection but have not yet been diagnosed with an flavivirusinfection.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a flavivirus infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of from about 25 mg to about 600 mg orally,subcutaneously, or intramuscularly bid, tid, qd, qod, tiw, or biw, orper day, substantially continuously or continuously, for the desiredtreatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a flavivirus infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of about 25 mg orally three times per day for thedesired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a flavivirus infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of about 50 mg orally three times per day for thedesired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a flavivirus infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of about 100 mg orally three times per day for thedesired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a flavivirus infection in a patient,comprising administering to the patient acarbose at a dosage of about 25mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a flavivirus infection in a patient,comprising administering to the patient acarbose at a dosage of about 50mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a flavivirus infection in a patient,comprising administering to the patient acarbose at a dosage of about100 mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a flavivirus infection in a patient,comprising administering to the patient miglitol at a dosage of about 25mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a flavivirus infection in a patient,comprising administering to the patient miglitol at a dosage of about 50mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a flavivirus infection in a patient,comprising administering to the patient miglitol at a dosage of about100 mg orally three times per day for the desired treatment duration.

Methods of Treating a Hepatitis C Virus Infection

The present invention provides monotherapy methods of treating an HCVinfection by administering to an individual in need thereof atherapeutically effective amount of an agent that inhibits enzymaticactivity of a membrane-bound α-glucosidase. Individuals who are to betreated according to the methods of the invention include individualswho have been clinically diagnosed with an HCV infection, as well asindividuals who exhibit one or more of the signs and the symptoms ofclinical infection but have not yet been diagnosed with an HCVinfection.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of an HCV infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of from about 30 mg to about 600 mg orally,subcutaneously, or intramuscularly bid, tid, qd, qod, tiw, or biw, orper day, substantially continuously or continuously, for the desiredtreatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of an HCV infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of about 25 mg orally three times per day for thedesired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of an HCV infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of about 50 mg orally three times per day for thedesired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of an HCV infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of about 100 mg orally three times per day for thedesired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of an HCV infection in a patient,comprising administering to the patient acarbose at a dosage of about 25mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of an HCV infection in a patient,comprising administering to the patient acarbose at a dosage of about 50mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of an HCV infection in a patient,comprising administering to the patient acarbose at a dosage of about100 mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of an HCV infection in a patient,comprising administering to the patient miglitol at a dosage of about 25mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of an HCV infection in a patient,comprising administering to the patient miglitol at a dosage of about 50mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of an HCV infection in a patient,comprising administering to the patient miglitol at a dosage of about100 mg orally three times per day for the desired treatment duration.

Methods of Treating a West Nile Virus Infection

The present invention provides monotherapy methods of treating a WNVinfection by administering to an individual in need thereof atherapeutically effective amount of an agent that inhibits enzymaticactivity of a membrane-bound α-glucosidase. Individuals who are to betreated according to the methods of the invention include individualswho have been clinically diagnosed with a WNV infection, as well asindividuals who exhibit one or more of the signs arid the symptoms ofclinical infection but have not yet been diagnosed with a WNV infection.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a WNV infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of from about 30 mg to about 600 mg orally,subcutaneously, or intramuscularly bid, tid, qd, qod, tiw, or biw, orper day, substantially continuously or continuously, for the desiredtreatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a WNV infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of about 25 mg orally three times per day for thedesired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a WNV infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of about 50 mg orally three times per day for thedesired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a WNV infection in a patient,comprising administering to the patient the membrane-bound α-glucosidaseinhibitor at a dosage of about 100 mg orally three times per day for thedesired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a WNV infection in a patient,comprising administering to the patient acarbose at a dosage of about 25mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a WNV infection in a patient,comprising administering to the patient acarbose at a dosage of about 50mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a WNV infection in a patient,comprising administering to the patient acarbose at a dosage of about100 mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a WNV infection in a patient,comprising administering to the patient miglitol at a dosage of about 25mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a WNV infection in a patient,comprising administering to the patient miglitol at a dosage of about 50mg orally three times per day for the desired treatment duration.

In some embodiments, the invention provides a monotherapy method usingan effective amount of an agent that inhibits a membrane-boundα-glucosidase in the treatment of a WNV infection in a patient,comprising administering to the patient miglitol at a dosage of about100 mg orally three times per day for the desired treatment duration.

Combination Therapies

As discussed above, the present invention provides combination therapymethods of treating alphavirus infections; methods of treatingflavivirus infections; methods of treating hepatitis C virus (HCV)infections; methods of treating West Nile virus (WNV) infection; methodsof reducing liver fibrosis; methods of increasing liver function in anindividual suffering from liver fibrosis; methods of reducing theincidence of complications associated with HCV and cirrhosis of theliver; and methods of reducing viral load, or reducing the time to viralclearance, or reducing morbidity or mortality in the clinical outcomes,in patients suffering from flavivirus infection. The methods generallyinvolve administering to an individual in need thereof an effectiveamount an α-glucosidase inhibitor and at least a second therapeuticagent. Suitable additional therapeutic agents include, but are notlimited to, a Type I interferon receptor agonist, a Type II interferonreceptor agonist; a Type III interferon receptor agonist; a nucleosideanalog; an NS3 inhibitor, an NS5B inhibitor, viramidine, and thymosin-α.

Alpha-Glucosidase Inhibitors

Suitable α-glucosidase inhibitors include any of the above-describedimino-sugars, including long-alkyl chain derivatives of imino sugars asdisclosed in U.S. Patent Publication No. 2004/0110795; inhibitors ofendoplasmic reticulum-associated α-glucosidases; inhibitors of membranebound α-glucosidase; miglitol (Glyset®), and active derivatives, andanalogs thereof; and acarbose (Precose®), and active derivatives, andanalogs thereof.

In many embodiments, an α-glucosidase inhibitor is administered for aperiod of about 1 day to about 7 days, or about 1 week to about 2 weeks,or about 2 weeks to about 3 weeks, or about 3 weeks to about 4 weeks, orabout 1 month to about 2 months, or about 3 months to about 4 months, orabout 4 months to about 6 months, or about 6 months to about 8 months,or about 8 months to about 12 months, or at least one year, and may beadministered over longer periods of time.

An α-glucosidase inhibitor can be administered 5 times per day, 4 timesper day, tid (three times daily), bid, qd, qod, biw, tiw, qw, qow, threetimes per month, or once monthly. In other embodiments, an α-glucosidaseinhibitor is administered as a continuous infusion.

In many embodiments, an α-glucosidase inhibitor is administered orally.

In connection with the above-described methods for the treatment of aflavivirus infection, treatment of HCV infection, treatment of WNVinfection, and treatment of liver fibrosis that occurs as a result of anHCV infection, an α-glucosidase inhibitor is administered to the patientat a dosage of from about 10 mg per day to about 600 mg per day individed doses, e.g., from about 10 mg per day to about 30 mg per day,from about 30 mg per day to about 60 mg per day, from about 60 mg perday to about 75 mg per day, from about 75 mg per day to about 90 mg perday, from about 90 mg per day to about 120 mg per day, from about 120 mgper day to about 150 mg per day, from about 150 mg per day to about 180mg per day, from about 180 mg per day to about 210 mg per day, fromabout 210 mg per day to about 240 mg per day, from about 240 mg per dayto about 270 mg per day, from about 270 mg per day to about 300 mg perday, from about 300 mg per day to about 360 mg per day, from about 360mg per day to about 420 mg per day, from about 420 mg per day to about480 mg per day, or from about 480 mg to about 600 mg per day.

In some embodiments, an α-glucosidase inhibitor is administered in adosage of about 10 mg three times daily. In some embodiments, anα-glucosidase inhibitor is administered in a dosage of about 15 mg threetimes daily. In some embodiments, an α-glucosidase inhibitor isadministered in a dosage of about 20 mg three times daily. In someembodiments, an α-glucosidase inhibitor is administered in a dosage ofabout 25 mg three times daily. In some embodiments, an α-glucosidaseinhibitor is administered in a dosage of about 30 mg three times daily.In some embodiments, an α-glucosidase inhibitor is administered in adosage of about 40 mg three times daily. In some embodiments, anα-glucosidase inhibitor is administered in a dosage of about 50 mg threetimes daily. In some embodiments, an α-glucosidase inhibitor isadministered in a dosage of about 100 mg three times daily. In someembodiments, an α-glucosidase inhibitor is administered in a dosage ofabout 75 mg per day to about 150 mg per day in two or three divideddoses, where the individual weighs 60 kg or less. In some embodiments,an α-glucosidase inhibitor is administered in a dosage of about 75 mgper day to about 300 mg per day in two or three divided doses, where theindividual weighs 60 kg or more.

The amount of active ingredient (e.g., α-glucosidase inhibitor) that maybe combined with carrier materials to produce a dosage form can varydepending on the host to be treated and the particular mode ofadministration. A typical pharmaceutical preparation can contain fromabout 5% to about 95% active ingredient (w/w). In other embodiments, thepharmaceutical preparation can contain from about 20% to about 80%active ingredient.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific α-glucosidase inhibitor, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given α-glucosidase inhibitor are readilydeterminable by those of skill in the art by a variety of means. Atypical means is to measure the physiological potency of a given activeagent.

In many embodiments, multiple doses of an α-glucosidase inhibitor areadministered. For example, an α-glucosidase inhibitor is administeredonce per month, twice per month, three times per month, every other week(qow), once per week (qw), twice per week (biw), three times per week(tiw), four times per week, five times per week, six times per week,every other day (qod), daily (qd), twice a day (qid), or three times aday (tid), over a period of time ranging from about one day to about oneweek, from about two weeks to about four weeks, from about one month toabout two months, from about two months to about four months, from aboutfour months to about six months, from about six months to about eightmonths, from about eight months to about 1 year, from about 1 year toabout 2 years, or from about 2 years to about 4 years, or more.

Treatment Methods

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor in thetreatment of a flavivirus infection in a patient; and ii) at least oneadditional therapeutic agent in the treatment of a flavivirus infection(e.g., in the treatment of an HCV infection, in the treatment of a WNVinfection, etc.), as well as in a method of reducing liver fibrosis,etc., in a patient. The method generally comprises co-administering tothe patient a) a dosage of the α-glucosidase inhibitor; and b) a dosageof the at least one additional therapeutic agent for the desiredtreatment duration, to treat the flavivirus infection, reduce liverfibrosis, etc. In many embodiments, the α-glucosidase inhibitor isacarbose. In many embodiments, the α-glucosidase inhibitor is miglitol.

For convenience, the combination therapy treatment methods discussedbelow refer to “treating a flavivirus infection”; however, eachcombination therapy method is equally applicable to reducing liverfibrosis, reducing the incidence of liver cirrhosis, etc., as discussedabove. Moreover, each of the combination therapy treatment methodsdiscussed below that refer to “treating a flavivirus infection” areequally applicable to treating an HCV infection, e.g.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 10 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 15 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 20 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 25 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 30 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 35 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 40 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 50 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 60 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 70 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 75 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of acarbose containing anamount of 100 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 10 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 15 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 20 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 25 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 30 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 35 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 40 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 50 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 60 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 70 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 75 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) at least one additional therapeutic agent in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of miglitol containing anamount of 100 mg, administered orally tid; and b) a dosage of at leastone additional therapeutic agent administered at the desired frequencyand for the desired treatment duration, to treat the flavivirusinfection.

Combination Therapies with Type I Interferon Receptor Agonist

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor in thetreatment of a flavivirus infection in a patient; and ii) a Type Iinterferon receptor agonist and/or a Type III interferon receptoragonist in the treatment of a flavivirus infection in a patient. Themethod generally comprises co-administering to the patient a) a dosageof an α-glucosidase inhibitor; and b) a dosage of a Type I interferonreceptor agonist and/or a Type III interferon receptor agonist for thedesired treatment duration, to treat the flavivirus infection. In manyembodiments, the Type I interferon receptor agonist is an IFN-α. In manyembodiments, the α-glucosidase inhibitor is acarbose. In manyembodiments, the α-glucosidase inhibitor is miglitol. In manyembodiments, effective amounts of a Type I interferon receptor agonistand an α-glucosidase inhibitor are synergistic amounts.

Type I interferon receptor agonists suitable for use herein include anyinterferon-α (IFN-α). In certain embodiments, the interferon-α is aPEGylated interferon-α. In certain other embodiments, the interferon-αis a consensus interferon, such as INFERGEN® interferon alfacon-1. Instill other embodiments, the interferon-α is a monoPEG (30 kD,linear)-ylated consensus interferon.

Effective dosages of an IFN-α range from about 1 μg to about 3 μg, fromabout 3 μg to about 27 μg, from about 3 MU to about 10 MU, from about 90μg to about 180 μg, or from about 18 μg to about 90 μg. Effectivedosages of Infergen® consensus IFN-α include about 3 μg, about 6 μg,about 9 μg, about 12 μg, about 15 μg, about 18 μg, about 21 μg, about 24μg, about 27 μg, or about 30 μg, of drug per dose. Effective dosages ofIFN-α2a and IFN-α2b range from 3 million Units (MU) to 10 MU per dose.Effective dosages of PEGASYS®PEGylated IFN-α2a contain an amount ofabout 90 μg to 270 μg, or about 180 μg, of drug per dose. Effectivedosages of PEG-INTRON®PEGylated IFN-α2b contain an amount of about 0.5μg to 3.0 μg of drug per kg of body weight per dose. Effective dosagesof PEGylated consensus interferon (PEG-CIFN) contain an amount of about18 μg to about 90 μg, or from about 27 μg to about 60 μg, or about 45μg, of CIFN amino acid weight per dose of PEG-CIFN. Effective dosages ofmonoPEG (30 kD, linear)-ylated CIFN contain an amount of about 45 μg toabout 270 μg, or about 60 μg to about 180 μg, or about 90 μg to about120 μg, of drug per dose. IFN-α can be administered daily, every otherday, once a week, three times a week, every other week, three times permonth, once monthly, substantially continuously or continuously.

In many embodiments, the Type I or Type III interferon receptor agonistis administered for a period of about 1 day to about 7 days, or about 1week to about 2 weeks, or about 2 weeks to about 3 weeks, or about 3weeks to about 4 weeks, or about 1 month to about 2 months, or about 3months to about 4 months, or about 4 months to about 6 months, or about6 months to about 8 months, or about 8 months to about 12 months, or atleast one year, and may be administered over longer periods of time.Dosage regimens can include tid, bid, qd, qod, biw, tiw, qw, qow, threetimes per month, or monthly administrations. In some embodiments, theinvention provides any of the above-described methods in which thedesired dosage of IFN-α is administered subcutaneously to the patient bybolus delivery qd, qod, tiw, biw, qw, qow, three times per month, ormonthly, or is administered subcutaneously to the patient per day bysubstantially continuous or continuous delivery, for the desiredtreatment duration. In other embodiments, the invention provides any ofthe above-described methods in which the desired dosage of PEGylatedIFN-α (PEG-IFN-α) is administered subcutaneously to the patient by bolusdelivery qw, qow, three times per month, or monthly for the desiredtreatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType I interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) aType I interferon receptor agonist selected from: (i) INFERGEN®containing an amount of about 1 μg to about 30 μg of drug per dose ofINFERGEN® subcutaneously qd, qod, tiw, biw, qw, qow, three times permonth, once monthly, or per clay continuously or substantiallycontinuously (ii) PEGylated consensus IFN-α (PEG-CIFN) containing anamount of about 10 μg to about 100 μg, or about 40 μg to about 80 μg, ofCIFN amino acid weight per dose of PEG-CIFN subcutaneously qw, qow,three times per month, or monthly (iii) IFN-α 2a, 2b or 2c containing anamount of about 3 MU to about 10 MU of drug per dose of IFN-α 2a, 2b or2c subcutaneously qd, qod, tiw, biw, or per day continuously orsubstantially continuously (iv) PEGASYS® containing an amount of about90 μg to about 360 μg, or about 180 μg, of drug per dose of PEGASYS®subcutaneously qw, qow, three times per month, or monthly (v)PEG-INTRON® containing an amount of about 0.75 μg to about 3.0 μg, orabout 1.0 μg to about 1.5 μg, of drug per kilogram of body weight perdose of PEG-INTRON® subcutaneously biw, qw, qow, three times per month,or monthly or (vi) mono PEG(30 kD, linear)-ylated consensus IFN-αcontaining an amount of from about 100 μg to about 200 μg, or about 150μg, of drug per dose of mono PEG(30 kD, linear)-ylated consensus IFN-αsubcutaneously qw, qow, once every 8 days to once every 14 days, threetimes per month, or monthly for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType I interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) aType I interferon receptor agonist selected from: (i) INFERGEN®containing an amount of about 1 μg to about 30 μg of drug per dose ofINFERGEN® subcutaneously qd, qod, tiw, biw, qw, qow, three times permonth, once monthly, or per day continuously or substantiallycontinuously (ii) PEGylated consensus IFN-α (PEG-CIFN) containing anamount of about 10 μg to about 100 μg, or about 40 μg to about 80 μg, ofCIFN amino acid weight per dose of PEG-CIFN subcutaneously qw, qow,three times per month, or monthly (iii) IFN-α 2a, 2b or 2c containing anamount of about 3 MU to about 10 MU of drug per dose of IFN-α 2a, 2b or2c subcutaneously qd, qod, tiw, biw, or per day continuously orsubstantially continuously (iv) PEGASYS® containing an amount of about90 μg to about 360 μg, or about 180 μg, of drug per dose of PEGASYS®subcutaneously qw, qow, three times per month, or monthly (v)PEG-INTRON® containing an amount of about 0.75 μg to about 3.0 μg, orabout 1.0 μg to about 1.5 μg, of drug per kilogram of body weight perdose of PEG-INTRON® subcutaneously biw, qw, qow, three times per month,or monthly or (vi) mono PEG(30 kD, linear)-ylated consensus IFN-αcontaining an amount of from about 100 μg to about 200 μg, or about 150μg, of drug per dose of mono PEG(30 kD, linear)-ylated consensus IFN-αsubcutaneously qw, qow, once every 8 days to once every 14 days, threetimes per month, or monthly for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgmiglitol, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg miglitol,administered orally tid; and b) a dosage of INFERGEN® containing anamount of about 1 μg to about 30 μg e.g., 1 μg, 9 μg, 27 μg, or 30 μg ofdrug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow,three times per month, once monthly, or per day substantiallycontinuously or continuously for the desired treatment duration, totreat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgmiglitol, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg miglitol,administered orally tid; and b) a dosage of INFERGEN® consensus IFN-αcontaining an amount of from about 1 μg to about 30 μg, e.g., 1 μg, 18μg, 27 μg, or 30 μg INFERGEN® consensus IFN-α administeredsubcutaneously qd or tiw for the desired treatment duration, to treatthe flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgmiglitol, administered orally tid, e.g., 10 mg, 15 mg, 20 mg., 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg miglitol,administered orally tid; and b) a dosage of PEGylated consensus IFN-α(PEG-CIFN) containing an amount of about 18 μg to about 24 μg of CIFNamino acid weight per dose of PEG-CIFN, subcutaneously qw, qow, threetimes per month, or monthly for the desired treatment duration, to treatthe flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient: a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgmiglitol, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg miglitol,administered orally tid; and b) a dosage of PEGASYS® PEGylated IFN-α2acontaining an amount of about 90 μg to about 360 μg of drug per dose ofPEGASYS®, subcutaneously qw, qow, three times per month, or monthly forthe desired treatment duration, to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgmiglitol, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg miglitol,administered orally tid; and b) a dosage of PEGASYS® PEGylated IFN-α2acontaining an amount of about 180 μg of drug per dose of PEGASYS®,subcutaneously qw, qow, three times per month, or monthly for thedesired treatment duration, to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgmiglitol, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg miglitol,administered orally tid; and b) a dosage of PEG-INTRON® PEGylatedIFN-α2b containing an amount of about 0.75 μg to about 3.0 μg of drugper kilogram of body weight per dose of PEG-INTRON®, subcutaneously qw,qow, three times per month, or monthly for the desired treatmentduration, to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgmiglitol, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg miglitol,administered orally tid; and b) a dosage of PEG-INTRON® containing anamount of about 1.5 μg of drug per kilogram of body weight per dose ofPEG-INTRON®, subcutaneously qw, qow, three times per month, or monthlyfor the desired treatment duration, to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient: a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgmiglitol, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg miglitol,administered orally tid; and b) a dosage of IFN-α containing an amountof 100 μg rnonoPEG(30 kD, linear)-ylated consensus IFN-α administeredsubcutaneously every 10 days or qw for the desired treatment duration,to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgmiglitol, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg miglitol,administered orally tid; and b) a dosage of IFN-α containing an amountof 150 μg monoPEG(30 kD, linear)-ylated consensus IFN-α administeredsubcutaneously every 10 days or qw for the desired treatment duration,to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgmiglitol, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg miglitol,administered orally tid; and b) a dosage of IFN-α containing an amountof 200 μg monoPEG(30 kD, linear)-ylated consensus IFN-α administeredsubcutaneously every 10 days or qw for the desired treatment duration,to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgacarbose, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg acarbose,administered orally tid; and b) a dosage of INFERGEN® containing anamount of about 1 μg to about 30 μg, e.g., 1 μg, 9 μg, 27 μg, or 30 μgof drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw,qow, three times per month, once monthly, or per day substantiallycontinuously or continuously for the desired treatment duration, totreat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgacarbose, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg acarbose,administered orally tid; and b) a dosage of INFERGEN® consensus IFN-αcontaining an amount of from about 1 μg to about 30 μg, e.g., 1 μg, 9μg, 18 μg, 27 μg, or 30 μg INFERGEN® consensus IFN-α administeredsubcutaneously qd or tiw for the desired treatment duration, to treatthe flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgacarbose, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg acarbose,administered orally tid; and b) a dosage of PEGylated consensus IFN-α(PEG-CIFN) containing an amount of about 18 μg to about 24 μg of CIFNamino acid weight per dose of PEG-CIFN, subcutaneously qw, qow, threetimes per month, or monthly for the desired treatment duration, to treatthe flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgacarbose, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg acarbose,administered orally tid; and b) a dosage of PEGASYS® PEGylated IFN-α2acontaining an amount of about 90 μg to about 360 μg of drug per dose ofPEGASYS®, subcutaneously qw, qow, three times per month, or monthly forthe desired treatment duration, to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgacarbose, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg acarbose,administered orally tid; and b) a dosage of PEGASYS® PEGylated IFN-α2acontaining an amount of about 180 μg of drug per dose of PEGASYS®,subcutaneously qw, qow, three times per month, or monthly for thedesired treatment duration, to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgacarbose, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg acarbose,administered orally tid; and b) a dosage of PEG-INTRON® PEGylatedIFN-α2b containing an amount of about 0.75 μg to about 3.0 μg of drugper kilogram of body weight per dose of PEG-INTRON®, subcutaneously qw,qow, three times per month, or monthly for the desired treatmentduration, to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgacarbose, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg acarbose,administered orally tid; and b) a dosage of PEG-INTRON® containing anamount of about 1.5 μg of drug per kilogram of body weight per dose ofPEG-INTRON®, subcutaneously qw, qow, three times per month, or monthlyfor the desired treatment duration, to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgacarbose, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg acarbose,administered orally tid; and b) a dosage of IFN-α containing an amountof 100 μg monoPEG(30 kD, linear)-ylated consensus IFN-α administeredsubcutaneously every 10 days or qw for the desired treatment duration,to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgacarbose, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg acarbose,administered orally tid; and b) a dosage of IFN-α containing an amountof 150 μg monoPEG(30 kD, linear)-ylated consensus IFN-α administeredsubcutaneously every 10 days or qw for the desired treatment duration,to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor; andii) a Type I interferon receptor agonist in the treatment of aflavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of from about 10 mg to about 100 mgacarbose, administered orally tid, e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or 100 mg acarbose,administered orally tid; and b) a dosage of IFN-α containing an amountof 200 μg monoPEG(30 kD, linear)-ylated consensus IFN-α administeredsubcutaneously every 10 days or qw for the desired treatment duration,to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType I interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof 10 mg miglitol, administered orally tid; and b) administering adosage of INFERGEN® containing an amount of about 1 μg of drug per doseof INFERGEN® consensus IFN-α subcutaneously tiw, for the desiredtreatment duration.

In one non-limiting example, the invention provides a combinationtherapy method using combined effective amounts of an α-glucosidaseinhibitor; and a Type I interferon receptor agonist in the treatment ofa flavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of 10 mg miglitol, administered orallytid; and b) administering a dosage of INFERGEN® consensus IFN-αcontaining an amount of about 9 μg of drug per dose of INFERGEN®consensus IFN-α subcutaneously tiw, for the desired treatment duration.

In one non-limiting example, the invention provides a combinationtherapy method using combined effective amounts of an α-glucosidaseinhibitor; and a Type I interferon receptor agonist in the treatment ofa flavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of 25 mg miglitol, administered orallytid; and b) administering a dosage of INFERGEN® consensus IFN-αcontaining an amount of about 9 μg of drug per dose of INFERGEN®consensus IFN-α subcutaneously tiw, for the desired treatment duration.

In one non-limiting example, the invention provides a combinationtherapy method using combined effective amounts of an α-glucosidaseinhibitor; and a Type I interferon receptor agonist in the treatment ofa flavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of 10 mg acarbose, administered orallytid; and b) administering a dosage of INFERGEN® consensus IFN-αcontaining an amount of about 1 μg of drug per dose of INFERGEN®consensus IFN-α subcutaneously tiw, for the desired treatment duration.

In one non-limiting example, the invention provides a combinationtherapy method using combined effective amounts of an α-glucosidaseinhibitor; and a Type I interferon receptor agonist in the treatment ofa flavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of 10 mg acarbose, administered orallytid; and b) administering a dosage of INFERGEN® consensus IFN-αcontaining an amount of about 9 μg of drug per dose of INFERGEN®consensus IFN-α subcutaneously tiw, for the desired treatment duration.

In one non-limiting example, the invention provides a combinationtherapy method using combined effective amounts of an α-glucosidaseinhibitor; and a Type I interferon receptor agonist in the treatment ofa flavivirus infection in a patient, the method comprisingco-administering to the patient a) a dosage of an α-glucosidaseinhibitor containing an amount of 25 mg acarbose, administered orallytid; and b) administering a dosage of INFERGEN® consensus IFN-αcontaining an amount of about 9 μg of drug per dose of INFERGEN®consensus IFN-α subcutaneously tiw, for the desired treatment duration.

Combination Therapies with a Type II Interferon Receptor Agonist

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of i) an α-glucosidase inhibitor in thetreatment of a flavivirus infection in a patient; and ii) a Type IIinterferon receptor agonist in the treatment of a flavivirus infectionin a patient. The method generally comprises co-administering to thepatient a) a dosage of an α-glucosidase inhibitor; and b) a dosage of aType II interferon receptor agonist for the desired treatment duration,to treat the flavivirus infection. In many embodiments, the Type IIinterferon receptor agonist is an IFN-γ. In many embodiments, theα-glucosidase inhibitor is acarbose. In many embodiments, theα-glucosidase inhibitor is miglitol. In many embodiments, effectiveamounts of a Type II interferon receptor agonist and an α-glucosidaseinhibitor are synergistic amounts.

Effective dosages of IFN-γ can range from about 0.5 μg/m² to about 500μg/m², usually from about 1.5 μg/m² to 200 μg/m², depending on the sizeof the patient. This activity is based on 10⁶ international units (U)per 50 μg of protein. IFN-γ can be administered daily, every other day,three times a week (tiw), or substantially continuously or continuously.In specific embodiments of interest, IFN-γ is administered to anindividual in a unit dosage form of from about 25 μg to about 500 μg,from about 50 μg to about 400 μg, or from about 100 μg to about 300 μg.In particular embodiments of interest, the dose is about 200 μg IFN-γ.In many embodiments of interest, IFN-γ1b is administered. In someembodiments, the IFN-γ is Actimmune® human IFN-γ1b.

Where the dosage is 200 μg IFN-γ per dose, the amount of IFN-γ per bodyweight (assuming a range of body weights of from about 45 kg to about135 kg) is in the range of from about 4.4 μg IFN-γ per kg body weight toabout 1.48 μg IFN-γ per kg body weight.

The body surface area of individuals to be treated generally ranges fromabout 1.33 m² to about 2.50 m². Thus, in many embodiments, an IFN-γdosage ranges from about 150 μg/m² to about 20 μg/m². For example, anIFN-γ dosage ranges from about 20 μg/m² to about 30 μg/m², from about 30μg/m² to about 40 μg/m², from about 40 μg/m² to about 50 μg/m², fromabout 50 μg/m² to about 60 μg/m², from about 60 μg/m² to about 70 μg/m²,from about 70 μg/m² to about 80 μg/m², from about 80 μg/m² to about 90μg/m², from about 90 μg/m² to about 100 μg/m², from about 100 μg/m² toabout 110 μg/m², from about 110 μg/m² to about 120 μg/m², from about 120μg/m² to about 130 μg/m², from about 130 μg/m² to about 140 μg/m², orfrom about 140 μg/m² to about 150 μg/m². In some embodiments, the dosagegroups range from about 25 μg/m² to about 100 μg/m². In otherembodiments, the dosage groups range from about 25 μg/m² to about 50μg/m².

In many embodiments, multiple doses of an IFN-γ are administered. Forexample, an IFN-γ is administered once per month, twice per month, threetimes per month, every other week (qow), once per week (qw), twice perweek (biw), three times per week (tiw), four times per week, five timesper week, six times per week, every other day (qod), daily (qd),substantially continuously, or continuously, over a period of timeranging from about one day to about one week, from about two weeks toabout four weeks, from about one month to about two months, from abouttwo months to about four months, from about four months to about sixmonths, from about six months to about eight months, from about eightmonths to about 1 year, from about 1 year to about 2 years, or fromabout 2 years to about 4 years, or more.

In some embodiments, the IFN-γ is Actimmune® human IFN-γ1b, and isadministered subcutaneously tiw in a dosage containing an amount ofabout 25 μg, 50 μg, 100 μg, 150 μg, or 200 μg.

In some embodiments, effective dosages of IFN-γ range from about 0.5μg/m² to about 500 μg/m², e.g., from about 1.5 μg/m² to 200 μg/m²,depending on the size of the patient. This activity is based on 10⁶international units (IU) per 50 μg of protein.

Where the agent is a polypeptide, polynucleotide (e.g., a polynucleotideencoding IFN-γ), it may be introduced into tissues or host cells by anynumber of routes, including viral infection, microinjection, or fusionof vesicles. Jet injection may also be used for intramuscularadministration, as described by Furth et al. (1992), Anal Biochem205:365-368. The DNA may be coated onto gold microparticles, anddelivered intradermally by a particle bombardment device, or “gene gun”as described in the literature (see, for example, Tang et al. (1992),Nature 356:152-154), where gold microprojectiles are coated with thetherapeutic DNA, then bombarded into skin cells. Of particular interestin these embodiments is use of a liver-specific promoter to drivetranscription of an operably linked IFN-γ coding sequence preferentiallyin liver cells.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

In particular embodiments of interest, IFN-γ is administered as asolution suitable for subcutaneous injection. For example, IFN-γ is in aformulation containing 40 mg mannitol/mL, 0.72 mg sodium succinate/mL,0.10 mg polysorbate 20/mL. In particular embodiments of interest, IFN-γis administered in single-dose forms of 200 μg/dose subcutaneously.

Multiple doses of IFN-γ can be administered, e.g., IFN-γ can beadministered once per month, twice per month, three times per month,once per week, twice per week, three times per week, four times perweek, five times per week, six times per week, or daily, over a periodof time ranging from about one day to about one week, from about twoweeks to about four weeks, from about one month to about two months,from about two months to about four months, from about four months toabout six months, from about six months to about eight months, fromabout eight months to about 1 year, from about 1 year to about 2 years,or from about 2 years to about 4 years, or more. In particularembodiments of interest, IFN-γ is administered three times per week overa period of about 48 weeks.

In some embodiments, a Type II interferon receptor agonist (e.g., IFN-γ)is administered throughout the entire course of the α-glucosidaseinhibitor treatment. In other embodiments, a Type II interferon receptoragonist is administered less than the entire course of α-glucosidaseinhibitor treatment, e.g., only during the first phase of α-glucosidaseinhibitor treatment, only during the second phase of α-glucosidaseinhibitor treatment, or some other portion of the α-glucosidaseinhibitor treatment regimen.

In some embodiments, the Type II interferon receptor agonist and theα-glucosidase inhibitor are administered in the same formulation, andare administered simultaneously. In other embodiments, the Type IIinterferon receptor agonist and the α-glucosidase inhibitor areadministered separately, e.g., in separate formulations. In some ofthese embodiments, the Type II interferon receptor agonist and theα-glucosidase inhibitor are administered separately, and areadministered simultaneously. In other embodiments, the Type IIinterferon receptor agonist and the α-glucosidase inhibitor areadministered separately and are administered within about 5 seconds toabout 15 seconds, within about 15 seconds to about 30 seconds, withinabout 30 seconds to about 60 seconds, within about 1 minute to about 5minutes, within about 5 minutes to about 15 minutes, within about 15minutes to about 30 minutes, within about 30 minutes to about 60minutes, within about 1 hour to about 2 hours, within about 2 hours toabout 6 hours, within about 6 hours to about 12 hours, within about 12hours to about 24 hours, or within about 24 hours to about 48 hours ofone another.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) asize-based dosage of IFN-γ containing an amount of from about 25 μg/m²to about 100 μg/m², or a fixed dosage of IFN-γ containing an amount offrom about 50 μg to about 200 μg, administered subcutaneously tiw forthe desired treatment duration, to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) asize-based dosage of IFN-γ containing an amount of from about 25 μg/m²to about 100 μg/m², or a fixed dosage of IFN-γ containing an amount offrom about 50 μg to about 200 μg, administered subcutaneously tiw forthe desired treatment duration, to treat the flavivirus infection.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) adosage of IFN-γ containing an amount of about 10 μg to about 300 μg ofdrug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, threetimes per month, once monthly, or per day substantially continuously orcontinuously, for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) adosage of IFN-γ containing an amount of about 10 μg to about 300 μg ofdrug per dose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, threetimes per month, once monthly, or per day substantially continuously orcontinuously, for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) adosage of IFN-γ containing an amount of about 25 μg IFN-γ administeredsubcutaneously tiw, for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) adosage of IFN-γ containing an amount of about 25 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, for the desired treatmentduration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) adosage of IFN-γ containing an amount of about 25 μg IFN-γ administeredsubcutaneously tiw, for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) adosage of IFN-γ containing an amount of about 25 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, for the desired treatmentduration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) adosage of IFN-γ containing an amount of about 50 μg IFN-γ administeredsubcutaneously tiw, for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) adosage of IFN-γ containing an amount of about 50 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, for the desired treatmentduration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) adosage of IFN-γ containing an amount of about 50 μg IFN-γ administeredsubcutaneously tiw, for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) adosage of IFN-γ containing an amount of about 50 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, for the desired treatmentduration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) adosage of IFN-γ containing an amount of about 100 μg IFN-γ administeredsubcutaneously tiw, for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 rag, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) adosage of IFN-γ containing an amount of about 100 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, for the desired treatmentduration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) adosage of IFN-γ containing an amount of about 100 μg IFN-γ administeredsubcutaneously tiw, for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) adosage of IFN-γ containing an amount of about 100 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, for the desired treatmentduration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) adosage of IFN-γ containing an amount of about 200 μg IFN-γ administeredsubcutaneously tiw, for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg miglitol, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg miglitol, administered orally tid; and b) adosage of IFN-γ containing an amount of about 200 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, for the desired treatmentduration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) adosage of IFN-γ containing an amount of about 200 μg IFN-γ administeredsubcutaneously tiw, for the desired treatment duration.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; and aType II interferon receptor agonist in the treatment of a flavivirusinfection in a patient, the method comprising co-administering to thepatient a) a dosage of an α-glucosidase inhibitor containing an amountof from about 10 mg to about 100 mg acarbose, administered orally tid,e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75mg, 80 mg, 90 mg, or 100 mg acarbose, administered orally tid; and b) adosage of IFN-γ containing an amount of about 200 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, for the desired treatmentduration.

Combinations using an α-Glucosidase Inhibitor, a Type I InterferonReceptor Agonist, and a Type II Interferon Receptor Agonist

In some embodiments, the invention provides methods using an amount of aType I or Type III interferon receptor agonist, a Type II interferonreceptor agonist, and a α-glucosidase inhibitor, effective for thetreatment of a flavivirus in a patient. In some embodiments, theinvention provides methods using an effective amount of an IFN-α, IFN-γ,and an α-glucosidase inhibitor in the treatment of a flavivirusinfection in a patient. In one embodiment, the invention provides amethod using an effective amount of a consensus IFN-α, IFN-γ andα-glucosidase inhibitor in the treatment of a flavivirus in a patient.

In some embodiments, a Type I or a Type III interferon receptor agonistis administered in a first dosing regimen, followed by a second dosingregimen. The first dosing regimen of Type I or a Type III interferonreceptor agonist (also referred to as “the induction regimen”) generallyinvolves administration of a higher dosage of the Type I or Type IIIinterferon receptor agonist. For example, in the case of Infergen®consensus IFN-α (CIFN), the first dosing regimen comprises administeringCIFN at about 9 μg, about 15 μg, about 18 μg, or about 27 μg. The firstdosing regimen can encompass a single dosing event, or at least two ormore dosing events. The first dosing regimen of the Type I or Type IIIinterferon receptor agonist can be administered daily, every other day,three times a week, every other week, three times per month, oncemonthly, substantially continuously or continuously.

The first dosing regimen of the Type I or Type III interferon receptoragonist is administered for a first period of time, which time periodcan be at least about 4 weeks, at least about 8 weeks, or at least about12 weeks.

The second dosing regimen of the Type I or Type III interferon receptoragonist (also referred to as “the maintenance dose”) generally involvesadministration of a lower amount of the Type I or Type III interferonreceptor agonist. For example, in the case of CIFN, the second dosingregimen comprises administering CIFN at a dose of at least about 3 μg,at least about 9 μg, at least about 15 μg, or at least about 18 μg. Thesecond dosing regimen can encompass a single dosing event, or at leasttwo or more dosing events.

The second dosing regimen of the Type I or Type III interferon receptoragonist can be administered daily, every other day, three times a week,every other week, three times per month, once monthly, substantiallycontinuously or continuously.

In some embodiments, where an “induction”/“maintenance” dosing regimenof a Type I or a Type III interferon receptor agonist is administered, a“priming” dose of a Type II interferon receptor agonist (e.g., IFN-γ) isincluded. In these embodiments, IFN-γ is administered for a period oftime from about 1 day to about 14 days, from about 2 days to about 10days, or from about 3 days to about 7 days, before the beginning oftreatment with the Type I or Type III interferon receptor agonist. Thisperiod of time is referred to as the “priming” phase.

In some of these embodiments, the Type II interferon receptor agonisttreatment is continued throughout the entire period of treatment withthe Type I or Type III interferon receptor agonist. In otherembodiments, the Type II interferon receptor agonist treatment isdiscontinued before the end of treatment with the Type I or Type IIIinterferon receptor agonist. In these embodiments, the total time oftreatment with Type II interferon receptor agonist (including the“priming” phase) is from about 2 days to about 30 days, from about 4days to about 25 days, from about 8 days to about 20 days, from about 10days to about 18 days, or from about 12 days to about 16 days. In stillother embodiments, the Type II interferon receptor agonist treatment isdiscontinued once Type I or a Type III interferon receptor agonisttreatment begins.

In other embodiments, the Type I or Type interferon receptor agonist isadministered in single dosing regimen. For example, in the case of CIFN,the dose of CIFN is generally in a range of from about 3 μg to about 15μg, or from about 9 μg to about 15 μg. The dose of Type I or a Type IIIinterferon receptor agonist is generally administered daily, every otherday, three times a week, every other week, three times per month, oncemonthly, or substantially continuously. The dose of the Type I or TypeIII interferon receptor agonist is administered for a period of time,which period can be, for example, from at least about 24 weeks to atleast about 48 weeks, or longer.

In some embodiments, where a single dosing regimen of a Type I or a TypeIII interferon receptor agonist is administered, a “priming” dose of aType II interferon receptor agonist (e.g., IFN-γ) is included. In theseembodiments, IFN-γ is administered for a period of time from about 1 dayto about 14 days, from about 2 days to about 10 days, or from about 3days to about 7 days, before the beginning of treatment with the Type Ior Type III interferon receptor agonist. This period of time is referredto as the “priming” phase. In some of these embodiments, the Type IIinterferon receptor agonist treatment is continued throughout the entireperiod of treatment with the Type I or Type III interferon receptoragonist. In other embodiments, the Type II interferon receptor agonisttreatment is discontinued before the end of treatment with the Type I orType III interferon receptor agonist. In these embodiments, the totaltime of treatment with the Type II interferon receptor agonist(including the “priming” phase) is from about 2 days to about 30 days,from about 4 days to about 25 days, from about 8 days to about 20 days,from about 10 days to about 18 days, or from about 12 days to about 16days. In still other embodiments, Type II interferon receptor agonisttreatment is discontinued once Type I or a Type III interferon receptoragonist treatment begins.

In additional embodiments, an α-glucosidase inhibitor, a Type I or IIIinterferon receptor agonist, and a Type II interferon receptor agonistare co-administered for the desired duration of treatment in the methodsof the invention. In some embodiments, an α-glucosidase inhibitor, aninterferon-α, and an interferon-γ are co-administered for the desiredduration of treatment in the methods of the invention.

In general, an effective amount of a consensus interferon (CIFN) andIFN-γ suitable for use in the methods of the invention is provided by adosage ratio of 1 μg CIFN: 10 μg IFN-γ, where in some embodiments, bothCIFN and IFN-γ are unPEGylated and unglycosylated species.

In one embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of INFERGEN®consensus IFN-αand IFN-γ in the treatment of a flavivirus infection in a patientcomprising administering to the patient an α-glucosidase inhibitor; adosage of INFERGEN® containing an amount of about 1 μg to about 30 μg,of drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw,qow, three times per month, once monthly, or per day substantiallycontinuously or continuously; and a dosage of IFN-γ containing an amountof about 10 μg to about 300 μg of drug per dose of IFN-γ, subcutaneouslyqd, qod, tiw, biw, qw, qow, three times per month, once monthly, or perday substantially continuously or continuously, for the desired durationof treatment with the α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of INFERGEN®consensus IFN-αand IFN-γ in the treatment of a flavivirus infection in a patientcomprising administering to the patient a dosage of an α-glucosidaseinhibitor; and a dosage of INFERGEN® containing an amount of about 1 μgto about 9 μg, of drug per dose of INFERGEN®, subcutaneously qd, qod,tiw, biw, qw, qow, three times per month, once monthly, or per daysubstantially continuously or continuously; in combination with a dosageof IFN-γ containing an amount of about 10 μg to about 100 μg of drug perdose of IFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three timesper month, once monthly, or per day substantially continuously orcontinuously, for the desired duration of treatment with theα-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of INFERGEN®consensus IFN-αand IFN-γ in the treatment of a flavivirus infection in a patientcomprising administering to the patient a dosage of an α-glucosidaseinhibitor; and a dosage of INFERGEN® containing an amount of about 1 μgof drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw,qow, three times per month, once monthly, or per day substantiallycontinuously or continuously, in combination with a dosage of IFN-γcontaining an amount of about 10 μg to about 50 μg of drug per dose ofIFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month,once monthly, or per day substantially continuously or continuously, forthe desired duration of treatment with the α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of INFERGEN®consensus IFN-αand IFN-γ in the treatment of a flavivirus infection in a patientcomprising administering to the patient a dosage of an α-glucosidaseinhibitor; and a dosage of INFERGEN® containing an amount of about 9 μgof drug per dose of INFERGEN®, subcutaneously qd, qod, tiw, biw, qw,qow, three times per month, once monthly, or per day substantiallycontinuously or continuously, in combination with a dosage of IFN-γcontaining an amount of about 90 μg to about 100 μg of drug per dose ofIFN-γ, subcutaneously qd, qod, tiw, biw, qw, qow, three times per month,once monthly, or per day substantially continuously or continuously, forthe desired duration of treatment with the α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of INFERGEN®consensus IFN-αand IFN-γ in the treatment of a flavivirus infection in a patientcomprising administering to the patient a dosage of an α-glucosidaseinhibitor; and a dosage of an α-glucosidase inhibitor; and a dosage ofINFERGEN® containing an amount of about 30 μg of drug per dose ofINFERGEN®, subcutaneously qd, qod, tiw, biw, qw, qow, three times permonth, once monthly, or per day substantially continuously orcontinuously, in combination with a dosage of IFN-γ containing an amountof about 200 μg to about 300 μg of drug per dose of IFN-γ,subcutaneously qd, qod, tiw, biw, qw, qow, three times per month, oncemonthly, or per day substantially continuously or continuously, for thedesired duration of treatment with the α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of PEGylated consensus IFN-αand IFN-γ in the treatment of a flavivirus infection in a patientcomprising administering to the patient a dosage of an α-glucosidaseinhibitor; and a dosage of PEGylated consensus IFN-α (PEG-CIFN)containing an amount of about 4 μg to about 60 μg of CIFN amino acidweight per dose of PEG-CIFN, subcutaneously qw, qow, three times permonth, or monthly, in combination with a total weekly dosage of IFN-γcontaining an amount of about 30 μg to about 1,000 μg of drug per weekin divided doses administered subcutaneously qd, qod, tiw, biw, oradministered substantially continuously or continuously, for the desiredduration of treatment with the α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of PEGylated consensus IFN-αand IFN-γ in the treatment of a flavivirus infection in a patientcomprising administering to the patient a dosage of an α-glucosidaseinhibitor; and a dosage of PEGylated consensus IFN-α (PEG-CIFN)containing an amount of about 18 μg to about 24 μg of CIFN amino acidweight per dose of PEG-CIFN, subcutaneously qw, qow, three times permonth, or monthly, in combination with a total weekly dosage of IFN-γcontaining an amount of about 100 μg to about 300 μg of drug per week individed doses administered subcutaneously qd, qod, tiw, biw, orsubstantially continuously or continuously, for the desired duration oftreatment with the α-glucosidase inhibitor.

In general, an effective amount of IFN-α 2a or 2b or 2c and IFN-γsuitable for use in the methods of the invention is provided by a dosageratio of 1 million Units (MU) IFN-α 2a or 2b or 2c: 30 μg IFN-γ, whereboth IFN-α 2a or 2b or 2c and IFN-γ are unPEGylated and unglycosylatedspecies.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of IFN-α 2a or 2b or 2c andIFN-γ in the treatment of a flavivirus infection in a patient comprisingadministering to the patient a dosage of an α-glucosidase inhibitor; anda dosage of IFN-α 2a, 2b or 2c containing an amount of about 1 MU toabout 20 MU of drug per dose of IFN-α 2a, 2b or 2c subcutaneously qd,qod, tiw, biw, or per day substantially continuously or continuously, incombination with a dosage of IFN-γ containing an amount of about 30 μgto about 600 μg of drug per dose of IFN-γ, subcutaneously qd, qod, tiw,biw, or per day substantially continuously or continuously, for thedesired duration of treatment with the α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of IFN-α 2a or 2b or 2c andIFN-γ in the treatment of a flavivirus infection in a patient comprisingadministering to the patient a dosage of an α-glucosidase inhibitor;arid a dosage of IFN-α 2a, 2b or 2c containing an amount of about 3 MUof drug per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw,or per day substantially continuously or continuously, in combinationwith a dosage of IFN-γ containing an amount of about 100 μg of drug perdose of IFN-γ, subcutaneously qd, qod, tiw, biw, or per daysubstantially continuously or continuously, for the desired duration oftreatment with the α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of IFN-α 2a or 2b or 2c andIFN-γ in the treatment of a flavivirus infection in a patient comprisingadministering to the patient a dosage of an α-glucosidase inhibitor; anda dosage of IFN-α 2a, 2b or 2c containing an amount of about 10 MU ofdrug per dose of IFN-α 2a, 2b or 2c subcutaneously qd, qod, tiw, biw, orper day substantially continuously or continuously, in combination witha dosage of IFN-γ containing an amount of about 300 μg of drug per doseof IFN-γ, subcutaneously qd, qod, tiw, biw, or per day substantiallycontinuously or continuously, for the desired duration of treatment withthe α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of PEGASYS®PEGylated IFN-α2aand IFN-γ in the treatment of a flavivirus infection in a patientcomprising administering to the patient a dosage of an α-glucosidaseinhibitor; and a dosage of PEGASYS® containing an amount of about 90 μgto about 360 μg of drug per dose of PEGASYS®, subcutaneously qw, qow,three times per month, or monthly, in combination with a total weeklydosage of IFN-γ containing an amount of about 30 μg to about 1,000 μg ofdrug per week administered in divided doses subcutaneously qd, qod, tiw,or biw, or administered substantially continuously or continuously, forthe desired duration of treatment with the α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of PEGASYS®PEGylated IFN-α2aand IFN-γ in the treatment of a flavivirus infection in a patientcomprising administering to the patient a dosage of an α-glucosidaseinhibitor; and a dosage of PEGASYS® containing an amount of about 180 μgof drug per dose of PEGASYS®, subcutaneously qw, qow, three times permonth, or monthly, in combination with a total weekly dosage of IFN-γcontaining an amount of about 100 μg to about 300 μg, of drug per weekadministered in divided doses subcutaneously qd, qod, tiw, or biw, oradministered substantially continuously or continuously, for the desiredduration of treatment with the α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of PEG-INTRON®PEGylatedIFN-α2b and IFN-γ in the treatment of a flavivirus infection in apatient comprising administering to the patient a dosage of anα-glucosidase inhibitor; and a dosage of PEG-INTRON® containing anamount of about 0.75 μg to about 3.0 μg of drug per kilogram of bodyweight per dose of PEG-INTRON®, subcutaneously qw, qow, three times permonth, or monthly, in combination with a total weekly dosage of IFN-γcontaining an amount of about 30 μg to about 1,000 μg of drug per weekadministered in divided doses subcutaneously qd, qod, tiw, or biw, oradministered substantially continuously or continuously, for the desiredduration of treatment with the α-glucosidase inhibitor.

In another embodiment, the invention provides any of the above-describedmethods modified to use an effective amount of PEG-INTRON®PEGylatedIFN-α2b and IFN-γ in the treatment of a flavivirus infection in apatient comprising administering to the patient a dosage of anα-glucosidase inhibitor; and a dosage of PEG-INTRON® containing anamount of about 1.5 μg of drug per kilogram of body weight per dose ofPEG-INTRON®, subcutaneously qw, qow, three times per month, or monthly,in combination with a total weekly dosage of IFN-γ containing an amountof about 100 μg to about 300 μg of drug per week administered in divideddoses subcutaneously qd, qod, tiw, or biw, or administered substantiallycontinuously or continuously, for the desired duration of treatment withthe α-glucosidase inhibitor.

In one embodiment, the present invention provides,any of theabove-described methods modified to comprise administering to anindividual having a flavivirus infection an effective amount of anα-glucosidase inhibitor; and a regimen of 1 μg, 3 μg, or 9 μg INFERGEN®consensus IFN-α administered subcutaneously qd or tiw, and ribavirinadministered orally qd, where the duration of therapy is 48 weeks. Inthis embodiment, ribavirin is administered in an amount of 1000 mg forindividuals weighing less than 75 kg, and 1200 mg for individualsweighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 9 μg INFERGEN® consensus IFN-αadministered subcutaneously qd or tiw; 50 μg Actimmune Actimmune® humanIFN-γ1b administered subcutaneously tiw; and ribavirin administeredorally qd, where the duration of therapy is 48 weeks. In thisembodiment, ribavirin is administered in an amount of 1000 mg forindividuals weighing less than 75 kg, and 1200 mg for individualsweighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 9 μg INFERGEN® consensus IFN-αadministered subcutaneously qd or tiw; 100 μg Actimmune® human IFN-α1badministered subcutaneously tiw; and ribavirin administered orally qd,where the duration of therapy is 48 weeks. In this embodiment, ribavirinis administered in an amount of 1000 mg for individuals weighing lessthan 75 kg, and 1200 mg for individuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 9 μg INFERGEN® consensus IFN-αadministered subcutaneously qd or tiw; and 50 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, where the duration of therapyis 48 weeks.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 9 μg INFERGEN® consensus IFN-αadministered subcutaneously qd or tiw; and 100 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, where the duration of therapyis 48 weeks.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 9 μg INFERGEN® consensus IFN-αadministered subcutaneously qd or tiw; 25 μg Actimmune® human IFN-γ1badministered subcutaneously tiw; and ribavirin administered orally qd,where the duration of therapy is 48 weeks. In this embodiment, ribavirinis administered in an amount of 1000 mg for individuals weighing lessthan 75 kg, and 1200 mg for individuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 9 μg INFERGEN® consensus IFN-αadministered subcutaneously qd or tiw; 200 μg Actimmune® human IFN-γ1badministered subcutaneously tiw; and ribavirin administered orally qd,where the duration of therapy is 48 weeks. In this embodiment, ribavirinis administered in an amount of 1000 mg for individuals weighing lessthan 75 kg, and 1200 mg for individuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 9 μg INFERGEN® consensus IFN-αadministered subcutaneously qd or tiw; and 25 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, where the duration of therapyis 48 weeks.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 9 μg INFERGEN® consensus IFN-αadministered subcutaneously qd or tiw; and 200 μg Actimmune® humanIFN-γ1b administered subcutaneously tiw, where the duration of therapyis 48 weeks.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 100 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw, and ribavirin administered orally qd, where the duration oftherapy is 48 weeks. In this embodiment, ribavirin is administered in anamount of 1000 mg for individuals weighing less than 75 kg, and 1200 mgfor individuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 100 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; 50 μg Actimmune® human IFN-γ1b administered subcutaneously tiw;and ribavirin administered orally qd, where the duration of therapy is48 weeks. In this embodiment, ribavirin is administered in an amount of1000 mg for individuals weighing less than 75 kg, and 1200 mg forindividuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 100 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; 100 μg Actimmune® human IFN-γ1b administered subcutaneously tiw;and ribavirin administered orally qd, where the duration of therapy is48 weeks. In this embodiment, ribavirin is administered in an amount of1000 mg for individuals weighing less than 75 kg, and 1200 mg forindividuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 100 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; and 50 μg Actimmune® human IFN-γ1b administered subcutaneouslytiw, where the duration of therapy is 48 weeks.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 100 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; and 100 μg Actimmune® human IFN-γ1b administered subcutaneouslytiw, where the duration of therapy is 48 weeks.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 150 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw, and ribavirin administered orally qd, where the duration oftherapy is 48 weeks. In this embodiment, ribavirin is administered in anamount of 1000 mg for individuals weighing less than 75 kg, and 1200 mgfor individuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 150 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; 50 μg Actimmune® human. IFN-γ1b administered subcutaneously tiw;and ribavirin administered orally qd, where the duration of therapy is48 weeks. In this embodiment, ribavirin is administered in an amount of1000 mg for individuals weighing less than 75 kg, and 1200 mg forindividuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 150 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; 100 μg Actimmune® human IFN-γ1b administered subcutaneously tiw;and ribavirin administered orally qd, where the duration of therapy is48 weeks. In this embodiment, ribavirin is administered in an amount of1000 mg for individuals weighing less than 75 kg, and 1200 mg forindividuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 150 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; and 50 μg Actimmune® human IFN-γ1b administered subcutaneouslytiw, where the duration of therapy is 48 weeks.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 150 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; and 100 μg Actimmune® human IFN-γ1b administered subcutaneouslytiw, where the duration of therapy is 48 weeks.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 200 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw, and ribavirin administered orally qd, where the duration oftherapy is 48 weeks. In this embodiment, ribavirin is administered in anamount of 1000 mg for individuals weighing less than 75 kg, and 1200 mgfor individuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 200 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; 50 μg Actimmune® human IFN-γ1b administered subcutaneously tiw;and ribavirin administered orally qd, where the duration of therapy is48 weeks. In this embodiment, ribavirin is administered in an amount of1000 mg for individuals weighing less than 75 kg, and 1200 mg forindividuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 200 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; 100 μg Actimmune® human IFN-γ1b administered subcutaneously tiw;and ribavirin administered orally qd, where the duration of therapy is48 weeks. In this embodiment, ribavirin is administered in an amount of1000 mg for individuals weighing less than 75 kg, and 1200 mg forindividuals weighing 75 kg or more.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 200 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; and 50 μg Actimmune® human IFN-γ1b administered subcutaneouslytiw, where the duration of therapy is 48 weeks.

In one embodiment, the present invention provides any of theabove-described methods modified to comprise administering to anindividual having an HCV infection an effective amount of anα-glucosidase inhibitor; and a regimen of 200 μg monoPEG(30 kD,linear)-ylated consensus IFN-α administered subcutaneously every 10 daysor qw; and 100 μg Actimmune® human IFN-γ1b administered subcutaneouslytiw, where the duration of therapy is 48 weeks.

Combination Therapies with Ribavirin

In some embodiments, the methods provide for combination therapycomprising administering an α-glucosidase inhibitor, and an effectiveamount of ribavirin. Ribavirin can be administered in dosages of about400 mg, about 800 mg, about 1000 mg, or about 1200 mg per day.

In one embodiment, the invention provides any of the above-describedmethods modified to include co-administering to the patient atherapeutically effective amount of ribavirin for the duration of thedesired course of the α-glucosidase inhibitor treatment.

In another embodiment, the invention provides any of the above-describedmethods modified to include co-administering to the patient about 800 mgto about 1200 mg ribavirin orally per day for the duration of thedesired course of the α-glucosidase inhibitor treatment.

In another embodiment, the invention provides any of the above-describedmethods modified to include co-administering to the patient (a) 1000 mgribavirin orally per day if the patient has a body weight less than 75kg or (b) 1200 mg ribavirin orally per day if the patient has a bodyweight greater than or equal to 75 kg, where the daily dosage ofribavirin is optionally divided into to 2 doses for the duration of thedesired course of the α-glucosidase inhibitor treatment.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; andribavirin in the treatment of a flavivirus infection in a patient, themethod comprising co-administering to the patient a) a dosage of anα-glucosidase inhibitor containing an amount of from about 10 mg toabout 100 mg miglitol, administered orally tid, e.g., 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or100 mg miglitol, administered orally tid; and b) a dosage of ribavirincontaining an amount of from about 800 mg to about 1200 mg ribavirinorally per day for the duration of the desired course of theα-glucosidase inhibitor treatment.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; andribavirin in the treatment of a flavivirus infection in a patient, themethod comprising co-administering to the patient a) a dosage of anα-glucosidase inhibitor containing an amount of from about 10 mg toabout 100 mg acarbose, administered orally tid, e.g., 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or100 mg acarbose, administered orally tid; and b) a dosage of ribavirincontaining an amount of from about 800 mg to about 1200 mg ribavirinorally per day for the duration of the desired course of theα-glucosidase inhibitor treatment.

Combination Therapies with Levovirin

In some embodiments, the methods provide for combination therapycomprising administering an α-glucosidase inhibitor, as described above,and an effective amount of levovirin. Levovirin is generallyadministered in an amount ranging from about 30 mg to about 60 mg, fromabout 60 mg to about 125 mg, from about 125 mg to about 200 mg, fromabout 200 mg to about 300 mg, from about 300 mg to about 400 mg, fromabout 400 mg to about 1200 mg, from about 600 mg to about 1000 mg, orfrom about 700 to about 900 mg per day, or about 10 mg/kg body weightper day. In some embodiments, levovirin is administered orally indosages of about 400 mg, about 800 mg, about 1000 mg, or about 1200 mgper day for the desired course of the α-glucosidase treatment.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; andribavirin in the treatment of a flavivirus infection in a patient, themethod comprising co-administering to the patient a) a dosage of anα-glucosidase inhibitor containing an amount of from about 10 mg toabout 100 mg miglitol, administered orally tid, e.g., 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or100 mg miglitol, administered orally tid; and b) a dosage of levovirincontaining an amount of about 400 mg, 800 mg, 1000 mg, or about 1200 mgorally per day for the duration of the desired course of theα-glucosidase inhibitor treatment.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; andlevovirin in the treatment of a flavivirus infection in a patient, themethod comprising co-administering to the patient a) a dosage of anα-glucosidase inhibitor containing an amount of from about 10 mg toabout 100 mg acarbose, administered orally tid, e.g., 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or100 mg acarbose, administered orally tid; and b) a dosage of levovirincontaining an amount of about 400 mg, 800 mg, 1000 mg, or about 1200 mgorally per day for the duration of the desired course of theα-glucosidase inhibitor treatment.

Combination Therapies with Viramidine

In some embodiments, the methods provide for combination therapycomprising administering α-glucosidase inhibitor, as described above,and an effective amount of viramidine. Viramidine is generallyadministered in an amount ranging from about 30 mg to about 60 mg, fromabout 60 mg to about 125 mg, from about 125 mg to about 200 mg, fromabout 200 mg to about 300 mg, from about 300 mg to about 400 mg, fromabout 400 mg to about 1200 mg, from about 600 mg to about 1000 mg, orfrom about 700 to about 900 mg per day, or about 10 mg/kg body weightper day. In some embodiments, viramidine is administered orally indosages of about 800 mg, or about 1600 mg per day for the desired courseof the α-glucosidase inhibitor treatment.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; andviramidine in the treatment of a flavivirus infection in a patient, themethod comprising co-administering to the patient a) a dosage of anα-glucosidase inhibitor containing an amount of from about 10 mg toabout 100 mg miglitol, administered orally tid, e.g., 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or100 mg miglitol, administered orally tid; and b) a dosage of viramidinecontaining an amount of about 800 mg or about 1600 mg orally per day forthe duration of the desired course of the α-glucosidase inhibitortreatment.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; andviramidine in the treatment of a flavivirus infection in a patient, themethod comprising co-administering to the patient a) a dosage of anα-glucosidase inhibitor containing an amount of from about 10 mg toabout 100 mg acarbose, administered orally tid, e.g., 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or100 mg acarbose, administered orally tid; and b) a dosage of viramidinecontaining an amount of about 800 mg or about 1600 mg orally per day forthe duration of the desired course of the α-glucosidase inhibitortreatment.

Combination Therapies with Thymosin-α

In some embodiments, the methods provide for combination therapycomprising administering an α-glucosidase inhibitor, as described above,and an effective amount of thymosin-α. Thymosin-α (Zadaxin™) isgenerally administered by subcutaneous injection. Thymosin-α can beadministered tid, bid, qd, qod, biw, tiw, qw, qow, three times permonth, once monthly, substantially continuously, or continuously for thedesired course of the α-glucosidase inhibitor treatment. In manyembodiments, thymosin-α is administered twice per week for the desiredcourse of the α-glucosidase inhibitor treatment.

Effective dosages of thymosin-α range from about 0.5 mg to about 5 mg,e.g., from about 0.5 mg to about 1.0 mg, from about 1.0 mg to about 1.5mg, from about 1.5 mg to about 2.0 mg, from about 2.0 mg to about 2.5mg, from about 2.5 mg to about 3.0 mg, from about 3.0 mg to about 3.5mg, from about 3.5 mg to about 4.0 mg, from about 4.0 mg to about 4.5mg, or from about 4.5 mg to about 5.0 mg. In particular embodiments,thymosin-α is administered in dosages containing an amount of 1.0 mg or1.6 mg.

Thymosin-α can be administered over a period of time ranging from aboutone day to about one week, from about two weeks to about four weeks,from about one month to about two months, from about two months to aboutfour months, from about four months to about six months, from about sixmonths to about eight months, from about eight months to about 1 year,from about 1 year to about 2 years, or from about 2 years to about 4years, or more. In one emobidment, thymosin-α is administered for thedesired course of the α-glucosidase inhibitor treatment.

In one embodiment, the invention provides a method using an effectiveamount of ZADAXIN™ thymosin-α and an effective amount of an agent thatinhibits enzymatic activity of a membrane-bound α-glucosidase in thetreatment of a viral infection in a patient, comprising administering tothe patient a dosage of ZADAXIN™ containing an amount of from about 1.0mg to about 1.6 mg per dose, subcutaneously twice per week for thedesired duration of the α-glucosidase inhibitor treatment.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; andthymosin-α in the treatment of a flavivirus infection in a patient, themethod comprising co-administering to the patient a) a dosage of anα-glucosidase inhibitor containing an amount of from about 10 mg toabout 100 mg miglitol, administered orally tid, e.g., 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or100 mg miglitol, administered orally tid; and b) a dosage of thymosin-αcontaining an amount of from about 1.0 mg to about 1.6 mg per dose,subcutaneously twice per week for the desired duration of theα-glucosidase inhibitor treatment.

In some embodiments, the invention provides a combination therapy methodusing combined effective amounts of an α-glucosidase inhibitor; andthymosin-α in the treatment of a flavivirus infection in a patient, themethod comprising co-administering to the patient a) a dosage of anα-glucosidase inhibitor containing an amount of from about 10 mg toabout 100 mg acarbose, administered orally tid, e.g., 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, or100 mg acarbose, administered orally tid; and b) a dosage of thymosin-αcontaining an amount of from about 1.0 mg to about 1.6 mg per dose,subcutaneously twice per week for the desired duration of theα-glucosidase inhibitor treatment.

Combination Therapy with an HCV Enzyme Inhibitor

In some embodiments, a subject method provides for combination therapycomprising administering an effective amount of an α-glucosidaseinhibitor, as described above, and an effective amount of an HCV enzymeinhibitor, in combination therapy for treatment of an HCV infection. Insome of these embodiments, the methods provide for combination therapycomprising administering an effective amount of an α-glucosidaseinhibitor, as described above, and an effective amount of an NS3inhibitor, in combination therapy for treatment of an HCV infection. Insome of these embodiments, the methods provide for combination therapycomprising administering an effective amount of an α-glucosidaseinhibitor, as described above, and an effective amount of an NS5inhibitor, in combination therapy for treatment of an HCV infection.

In some embodiments, a subject therapeutic regimen involves modifyingany of the above-described regimens for HCV infection by administeringan HCV enzyme inhibitor. Effective dosages of an HCV enzyme inhibitorrange from about 10 mg to about 200 mg per dose, e.g., from about 10 mgto about 15 mg per dose, from about 15 mg to about 20 mg per dose, fromabout 20 mg to about 25 mg per dose, from about 25 mg to about 30 mg perdose, from about 30 mg to about 35 mg per dose, from about 35 mg toabout 40 mg per dose, from about 40 mg per dose to about 45 mg per dose,from about 45 mg per dose to about 50 mg per dose, from about 50 mg perdose to about 60 mg per dose, from about 60 mg per dose to about 70 mgper dose, from about 70 mg per dose to about 80 mg per dose, from about80 mg per dose to about 90 mg per dose, from about 90 mg per dose toabout 100 mg per dose, from about 100 mg per dose to about 125 mg perdose, from about 125 mg per dose to about 150 mg per dose, from about150 mg per dose to about 175 mg per dose, or from about 175 mg per doseto about 200 mg per dose.

In some embodiments, effective dosages of an HCV enzyme inhibitor areexpressed as mg/kg body weight. In these embodiments, effective dosagesof an HCV enzyme inhibitor are from about 0.01 mg/kg body weight toabout 100 mg/kg body weight, from about 0.1 mg/kg body weight to about50 mg/kg body weight, from about 0.1 mg/kg body weight to about 1 mg/kgbody weight, from about 1 mg/kg body weight to about 10 mg/kg bodyweigh, from about 10 mg/kg body weight to about 100 mg/kg body weight,from about 5 mg/kg body weight to about 400 mg/kg body weight, fromabout 5 mg/kg body weight to about 50 mg/kg body weight, from about 50mg/kg body weight to about 100 mg/kg body weight, from about 100 mg/kgbody weight to about 200 mg/kg body weight, from about 200 mg/kg bodyweight to about 300 mg/kg body weight, or from about 300 mg/kg bodyweight to about 400 mg/kg body weight.

In many embodiments, an HCV enzyme inhibitor is administered for aperiod of about 1 day to about 7 days, or about 1 week to about 2 weeks,or about 2 weeks to about 3 weeks, or about 3 weeks to about 4 weeks, orabout 1 month to about 2 months, or about 3 months to about 4 months, orabout 4 months to about 6 months, or about 6 months to about 8 months,or about 8 months to about 12 months, or at least one year, and may beadministered over longer periods of time. The HCV enzyme inhibitor canbe administered tid, bid, qd, qod, biw, tiw, qw, qow, three times permonth, once monthly, substantially continuously, or continuously.

In many embodiments, multiple doses of an HCV enzyme inhibitor areadministered. For example, an HCV enzyme inhibitor is administered onceper month, twice per month, three times per month, every other week(qow), once per week (qw), twice per week (biw), three times per week(tiw), four times per week, five times per week, six times per week,every other day (qod), daily (qd), twice a day (bid), or three times aday (tid), substantially continuously, or continuously, over a period oftime ranging from about one day to about one week, from about two weeksto about four weeks, from about one month to about two months, fromabout two months to about four months, from about four months to aboutsix months, from about six months to about eight months, from abouteight months to about 1 year, from about 1 year to about 2 years, orfrom about 2 years to about 4 years, or more.

As another example, in some embodiments, any of the above-describedtreatment regimens for treating an HCV infection is modified to includeadministering a dosage of an HCV NS3 protease inhibitor containing anamount of 0.01 mg to 100 mg of drug per kilogram of body weight orallydaily, optionally in two or more divided doses per day, for the desiredtreatment duration.

As another example, in some embodiments, any of the above-describedtreatment regimens for treating an HCV infection is modified to includeadministering a dosage of an HCV NS5B RNA-dependent RNA polymeraseinhibitor containing an amount of 0.01 mg to 100 mg of drug per kilogramof body weight orally daily, optionally in two or more divided doses perday, for the desired treatment duration.

As non-limiting examples, any of the above-described methods can bemodified to include an NS3 inhibitor regimen comprising administering adosage of 0.01 mg to 0.1 mg of drug per kilogram of body weight orallydaily, optionally in two or more divided doses per day, for the desiredtreatment duration.

As non-limiting examples, any of the above-described methods can bemodified to include an NS3 inhibitor regimen with an NS3 inhibitorregimen comprising administering a dosage of 0.1 mg to 1 mg of drug perkilogram of body weight orally daily, optionally in two or more divideddoses per day, for the desired treatment duration.

As non-limiting examples, any of the above-described methods can bemodified to include an NS3 inhibitor regimen with an NS3 inhibitorregimen comprising administering a dosage of 1 mg to 10 mg of drug perkilogram of body weight orally daily, optionally in two or more divideddoses per day, for the desired treatment duration.

As non-limiting examples, any of the above-described methods can bemodified to include an NS3 inhibitor regimen with an NS3 inhibitorregimen comprising administering a dosage of 10 mg to 100 mg of drug perkilogram of body weight orally daily, optionally in two or more divideddoses per day, for the desired treatment duration.

As non-limiting examples, any of the above-described methods can bemodified to include an NS5B inhibitor regimen comprising administering adosage of 0.01 mg to 0.1 mg of drug per kilogram of body weight orallydaily, optionally in two or more divided doses per day, for the desiredtreatment duration.

As non-limiting examples, any of the above-described methods can bemodified to include an NS5B inhibitor regimen with an NS5B inhibitorregimen comprising administering a dosage of 0.1 mg to 1 mg of drug perkilogram of body weight orally daily, optionally in two or more divideddoses per day, for the desired treatment duration.

As non-limiting examples, any of the above-described methods can bemodified to include an NS5B inhibitor regimen with an NS5B inhibitorregimen comprising administering a dosage of 1 mg to 10 mg of drug perkilogram of body weight orally daily, optionally in two or more divideddoses per day, for the desired treatment duration.

As non-limiting examples, any of the above-described methods can bemodified to include an NS5B inhibitor regimen with an NS5B inhibitorregimen comprising administering a dosage of 10 mg to 100 mg of drug perkilogram of body weight orally daily, optionally in two or more divideddoses per day, for the desired treatment duration.

Further Variations

As non-limiting examples, any of the above-described methods featuringan IFN-α regimen can be modified to replace the subject IFN-α regimenwith a regimen of monoPEG (30 kD, linear)-ylated consensus IFN-αcomprising administering a dosage of monoPEG (30 kD, linear)-ylatedconsensus IFN-α containing an amount of 50 μg of drug per dose,subcutaneously once weekly, once every 8 days, or once every 10 days forthe desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α regimen can be modified to replace the subject IFN-α regimenwith a regimen of monoPEG (30 kD, linear)-ylated consensus IFN-αcomprising administering a dosage of monoPEG (30 kD, linear)-ylatedconsensus IFN-α containing an amount of 100 μg of drug per dose,subcutaneously once weekly, once every 8 days, or once every 10 days forthe desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α regimen can be modified to replace the subject IFN-α regimenwith a regimen of monoPEG (30 kD, linear)-ylated consensus IFN-αcomprising administering a dosage of monoPEG (30 kD, linear)-ylatedconsensus IFN-α containing an amount of 150 μg of drug per dose,subcutaneously once weekly, once every 8 days, or once every 10 days forthe desired treatment.

As non-limiting examples, any of the above-described methods featuringan IFN-α regimen can be modified to replace the subject IFN-α regimenwith a regimen of monoPEG (30 kD, linear)-ylated consensus IFN-αcomprising administering a dosage of monoPEG (30 kD, linear)-ylatedconsensus IFN-α containing an amount of 200 μg of drug per dose,subcutaneously once weekly, once every 8 days, or once every 10 days forthe desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α regimen can be modified to replace the subject IFN-α regimenwith a regimen of INFERGEN® interferon alfacon-1 comprisingadministering a dosage of INFERGEN® interferon alfacon-1 containing anamount of 1 μg of drug per dose, subcutaneously once daily or threetimes per week for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α regimen can be modified to replace the subject IFN-α regimenwith a regimen of INFERGEN® interferon alfacon-1 comprisingadministering a dosage of INFERGEN® interferon alfacon-1 containing anamount of 3 μg of drug per dose, subcutaneously once daily or threetimes per week for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α regimen can be modified to replace the subject IFN-α regimenwith a regimen of INFERGEN® interferon alfacon-1 comprisingadministering a dosage of INFERGEN® interferon alfacon-1 containing anamount of 9 μg of drug per dose, subcutaneously once daily or threetimes per week for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α regimen can be modified to replace the subject IFN-α regimenwith a regimen of INFERGEN® interferon alfacon-1 comprisingadministering a dosage of INFERGEN® interferon alfacon-1 containing anamount of 15 μg of drug per dose, subcutaneously once daily or threetimes per week for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-γ regimen can be modified to replace the subject IFN-γ regimenwith a regimen of IFN-γ comprising administering a dosage of IFN-γcontaining an amount of 25 μg of drug per dose, subcutaneously threetimes per week for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-γ regimen can be modified to replace the subject IFN-γ regimenwith a regimen of IFN-γ comprising administering a dosage of IFN-γcontaining an amount of 50 μs of drug per dose, subcutaneously threetimes per week for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-γ regimen can be modified to replace the subject IFN-γ regimenwith a regimen of IFN-γ comprising administering a dosage of IFN-γcontaining an amount of 100 μg of drug per dose, subcutaneously threetimes per week for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of monoPEG(30 kD, linear)-ylated consensus IFN-α containing an amount of 50 μg ofdrug per dose, subcutaneously once weekly, once every 8 days, or onceevery 10 days; and (b) administering a dosage of IFN-γ containing anamount of 25 μg of drug per dose, subcutaneously three times per week;for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of monoPEG(30 kD, linear)-ylated consensus IFN-α containing an amount of 100 μg ofdrug per dose, subcutaneously once weekly, once every 8 days, or onceevery 10 days; and (b) administering a dosage of IFN-γ containing anamount of 50 μg of drug per dose, subcutaneously three times per week;for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of monoPEG(30 kD, linear)-ylated consensus IFN-α containing an amount of 100 μg ofdrug per dose, subcutaneously once weekly, once every 8 days, or onceevery 10 days; and (b) administering a dosage of IFN-γ containing anamount of 100 μg of drug per dose, subcutaneously three times per week;for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of monoPEG(30 kD, linear)-ylated consensus IFN-α containing an amount of 150 μg ofdrug per dose, subcutaneously once weekly, once every 8 days, or onceevery 10 days; and (b) administering a dosage of IFN-γ containing anamount of 50 μg of drug per dose, subcutaneously three times per week;for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of monoPEG(30 kD, linear)-ylated consensus IFN-α containing an amount of 150 μg ofdrug per dose, subcutaneously once weekly, once every 8 days, or onceevery 10 days; and (b) administering a dosage of IFN-γ containing anamount of 100 μg of drug per dose, subcutaneously three times per week;for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of monoPEG(30 kD, linear)-ylated consensus IFN-α containing an amount of 200 μg ofdrug per dose, subcutaneously once weekly, once every 8 days, or onceevery 10 days; and (b) administering a dosage of IFN-γ containing anamount of 50 μg of drug per dose, subcutaneously three times per week;for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of monoPEG(30 kD, linear)-ylated consensus IFN-α containing an amount of 200 μg ofdrug per dose, subcutaneously once weekly, once every 8 days, or onceevery 10 days; and (b) administering a dosage of IFN-γ containing anamount of 100 μg of drug per dose, subcutaneously three times per week;for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 9 μg of drug per dose,subcutaneously three times per week; and (b) administering a dosage ofIFN-γ containing an amount of 25 μg of drug per dose, subcutaneouslythree times per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 9 μg of drug per dose,subcutaneously three times per week; and (b) administering a dosage ofIFN-γ containing an amount of 50 μg of drug per dose, subcutaneouslythree times per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 9 μg of drug per dose,subcutaneously three times per week; and (b) administering a dosage ofIFN-γ containing an amount of 100 μg of drug per dose, subcutaneouslythree times per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 9 μg of drug per dose,subcutaneously once daily; and (b) administering a dosage of IFN-γcontaining an amount of 25 μg of drug per dose, subcutaneously threetimes per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 9 μg of drug per dose,subcutaneously once daily; and (b) administering a dosage of IFN-γcontaining an amount of 50 μg of drug per dose, subcutaneously threetimes per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 9 μg of drug per dose,subcutaneously once daily; and (b) administering a dosage of IFN-γcontaining an amount of 100 μg of drug per dose, subcutaneously threetimes per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 15 μg of drug per dose,subcutaneously three times per week; and (b) administering a dosage ofIFN-γ containing an amount of 25 μg of drug per dose, subcutaneouslythree times per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 15 μg of drug per dose,subcutaneously three times per week; and (b) administering a dosage ofIFN-γ containing an amount of 50 μg of drug per dose, subcutaneouslythree times per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 15 μg of drug per dose,subcutaneously three times per week; and (b) administering a dosage ofIFN-γ containing an amount of 100 μg of drug per dose, subcutaneouslythree times per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 15 μg of drug per dose,subcutaneously once daily; and (b) administering a dosage of IFN-γcontaining an amount of 25 μg of drug per dose, subcutaneously threetimes per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 15 μg of drug per dose,subcutaneously once daily; and (b) administering a dosage of IFN-γcontaining an amount of 50 μg of drug per dose, subcutaneously threetimes per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of INFERGEN®interferon alfacon-1 containing an amount of 15 μg of drug per dose,subcutaneously once daily; and (b) administering a dosage of IFN-γcontaining an amount of 100 μg of drug per dose, subcutaneously threetimes per week; for the desired treatment duration.

As non-limiting examples, any of the above-described methods featuringan IFN-α and IFN-γ combination regimen can be modified to replace thesubject IFN-α and IFN-γ combination regimen with an IFN-α and IFN-γcombination regimen comprising: (a) administering a dosage of monoPEG(30 kD, linear)-ylated consensus IFN-α containing an amount of 100 μg ofdrug per dose, subcutaneously once weekly, once every 8 days, or onceevery 10 days; and (b) administering a dosage of IFN-γ containing anamount of 100 μg of drug per dose, subcutaneously three times per weekfor the desired treatment duration.

As non-limiting examples, any of the above-described methods thatincludes a regimen of monoPEG (30 kD, linear)-ylated consensus IFN-α canbe modified to replace the regimen of monoPEG (30 kD, linear)-ylatedconsensus IFN-α with a regimen of peginterferon alfa-2a comprisingadministering a dosage of peginterferon alfa-2a containing an amount of180 μg of drug per dose, subcutaneously once weekly for the desiredtreatment duration.

As non-limiting examples, any of the above-described methods thatincludes a regimen of monoPEG (30 kD, linear)-ylated consensus IFN-α canbe modified to replace the regimen of monoPEG (30 kD, linear)-ylatedconsensus IFN-α with a regimen of peginterferon alfa-2b comprisingadministering a dosage of peginterferon alfa-2b containing an amount of1.0 μg to 1.5 μg of drug per kilogram of body weight per dose,subcutaneously once or twice weekly for the desired treatment duration.

As non-limiting examples, any of the above-described methods can bemodified to include administering a dosage of ribavirin containing anamount of 400 mg, 800 mg, 1000 mg or 1200 mg of drug orally per day,optionally in two or more divided doses per day, for the desiredtreatment duration.

As non-limiting examples, any of the above-described methods can bemodified to include administering a dosage of ribavirin containing (i)an amount of 1000 mg of drug orally per day for patients having a bodyweight of less than 75 kg or (ii) an amount of 1200 mg of drug orallyper day for patients having a body weight of greater than or equal to 75kg, optionally in two or more divided doses per day, for the desiredtreatment duration.

Combination Therapies with Other Antiviral Agents

Other antiviral agents are contemplated for use in combination therapiesdescribed herein. For example, ribozymes such as Heptazyme™ andphosphorothioate oligonucleotides which are complementary to HCV proteinsequences and which inhibit the expression of viral core proteins arealso suitable for use in combination therapies described herein.

In some embodiments, the additional antiviral agent(s) is administeredduring the entire course of treatment with an α-glucosidase inhibitor,and the beginning and end of the treatment periods coincide. In otherembodiments, the additional antiviral agent(s) is administered for aperiod of time that is overlapping with that of the α-glucosidaseinhibitor treatment, e.g., treatment with the additional antiviralagent(s) begins before the α-glucosidase inhibitor treatment begins andends before the α-glucosidase inhibitor treatment ends; treatment withthe additional antiviral agent(s) begins after the α-glucosidaseinhibitor treatment begins and ends after the α-glucosidase inhibitortreatment ends; treatment with the additional antiviral agent(s) beginsafter the α-glucosidase inhibitor treatment begins and ends before theα-glucosidase inhibitor treatment ends; or treatment with the additionalantiviral agent(s) begins before the α-glucosidase inhibitor treatmentbegins and ends after the α-glucosidase inhibitor treatment ends.

The α-glucosidase inhibitor can be administered together with (i.e.,simultaneously in separate formulations; simultaneously in the sameformulation; administered in separate formulations and within about 48hours, within about 36 hours, within about 24 hours, within about 16hours, within about 12 hours, within about 8 hours, within about 4hours, within about 2 hours, within about 1 hour, within about 30minutes, or within about 15 minutes or less) one or more additionalantiviral agents.

Patient Identification

In certain embodiments, the specific regimen of drug therapy used intreatment of the HCV patient is selected according to certain diseaseparameters exhibited by the patient, such as the initial viral load,genotype of the HCV infection in the patient, liver histology and/orstage of liver fibrosis in the patient.

Thus, in some embodiments, the present invention provides any of theabove-described methods for the treatment of HCV infection in which thesubject method is modified to treat a treatment failure patient for aduration of 48 weeks.

In other embodiments, the invention provides any of the above-describedmethods for HCV in which the subject method is modified to treat anon-responder patient, where the patient receives a 48 week course oftherapy.

In other embodiments, the invention provides any of the above-describedmethods for the treatment of HCV infection in which the subject methodis modified to treat a relapser patient, where the patient receives a 48week course of therapy.

In other embodiments, the invention provides any of the above-describedmethods for the treatment of HCV infection in which the subject methodis modified to treat a naïve patient infected with HCV genotype 1, wherethe patient receives a 48 week course of therapy.

In other embodiments, the invention provides any of the above-describedmethods for the treatment of HCV infection in which the subject methodis modified to treat a naïve patient infected with HCV genotype 4, wherethe patient receives a 48 week course of therapy.

In other embodiments, the invention provides any of the above-describedmethods for the treatment of HCV infection in which the subject methodis modified to treat a naïve patient infected with HCV genotype 1, wherethe patient has a high viral load (HVL), where “HVL” refers to an HCVviral load of greater than 2×10⁶ HCV genome copies per mL serum, andwhere the patient receives a 48 week course of therapy.

In one embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient havingadvanced or severe stage liver fibrosis as measured by a Knodell scoreof 3 or 4 and then (2) administering to the patient the drug therapy ofthe subject method for a time period of about 24 weeks to about 60weeks, or about 30 weeks to about one year, or about 36 weeks to about50 weeks, or about 40 weeks to about 48 weeks, or at least about 24weeks, or at least about 30 weeks, or at least about 36 weeks, or atleast about 40 weeks, or at least about 48 weeks, or at least about 60weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient havingadvanced or severe stage liver fibrosis as measured by a Knodell scoreof 3 or 4 and then (2) administering to the patient the drug therapy ofthe subject method for a time period of about 40 weeks to about 50weeks, or about 48 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 1 infection and an initial viral load of greater than 2million viral genome copies per ml of patient serum and then (2)administering to the patient the drug therapy of the subject method fora time period of about 24 weeks to about 60 weeks, or about 30 weeks toabout one year, or about 36 weeks to about 50 weeks, or about 40 weeksto about 48 weeks, or at least about 24 weeks, or at least about 30weeks, or at least about 36 weeks, or at least about 40 weeks, or atleast about 48 weeks, or at least about 60 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 1 infection and an initial viral load of greater than 2million viral genome copies per ml of patient serum and then (2)administering to the patient the drug therapy of the subject method fora time period of about 40 weeks to about 50 weeks, or about 48 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 1 infection and an initial viral load of greater than 2million viral genome copies per ml of patient serum and no or earlystage liver fibrosis as measured by a Knodell score of 0, 1, or 2 andthen (2) administering to the patient the drug therapy of the subjectmethod for a time period of about 24 weeks to about 60 weeks, or about30 weeks to about one year, or about 36 weeks to about 50 weeks, orabout 40 weeks to about 48 weeks, or at least about 24 weeks, or atleast about 30 weeks, or at least about 36 weeks, or at least about 40weeks, or at least about 48 weeks, or at least about 60 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 1 infection and an initial viral load of greater than 2million viral genome copies per ml of patient serum and no or earlystage liver fibrosis as measured by a Knodell score of 0, 1, or 2 andthen (2) administering to the patient the drug therapy of the subjectmethod for a time period of about 40 weeks to about 50 weeks, or about48 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 1 infection and an initial viral load of less than or equalto 2 million viral genome copies per ml of patient serum and then (2)administering to the patient the drug therapy of the subject method fora time period of about 20 weeks to about 50 weeks, or about 24 weeks toabout 48 weeks, or about 30 weeks to about 40 weeks, or up to about 20weeks, or up to about 24 weeks, or up to about 30 weeks, or up to about36 weeks, or up to about 48 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 1 infection and an initial viral load of less than or equalto 2 million viral genome copies per ml of patient serum and then (2)administering to the patient the drug therapy of the subject method fora time period of about 20 weeks to about 24 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 1 infection and an initial viral load of less than or equalto 2 million viral genome copies per ml of patient serum and then (2)administering to the patient the drug therapy of the subject method fora time period of about 24 weeks to about 48 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 2 or 3 infection and then (2) administering to the patientthe drug therapy of the subject method for a time period of about 24weeks to about 60 weeks, or about 30 weeks to about one year, or about36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or atleast about 24 weeks, or at least about 30 weeks, or at least about 36weeks, or at least about 40 weeks, or at least about 48 weeks, or atleast about 60 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 2 or 3 infection and then (2) administering to the patientthe drug therapy of the subject method for a time period of about 20weeks to about 50 weeks, or about 24 weeks to about 48 weeks, or about30 weeks to about 40 weeks, or up to about 20 weeks, or up to about 24weeks, or up to about 30 weeks, or up to about 36 weeks, or up to about48 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 2 or 3 infection and then (2) administering to the patientthe drug therapy of the subject method for a time period of about 20weeks to about 24 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 2 or 3 infection and then (2) administering to the patientthe drug therapy of the subject method for a time period of at leastabout 24 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV genotype 1 or 4 infection and then (2) administering to the patientthe drug therapy of the subject method for a time period of about 24weeks to about 60 weeks, or about 30 weeks to about one year, or about36 weeks to about 50 weeks, or about 40 weeks to about 48 weeks, or atleast about 24 weeks, or at least about 30 weeks, or at least about 36weeks, or at least about 40 weeks, or at least about 48 weeks, or atleast about 60 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having; anHCV infection characterized by any of HCV genotypes 5, 6, 7, 8 and 9 andthen (2) administering to the patient the drug therapy of the subjectmethod for a time period of about 20 weeks to about 50 weeks.

In another embodiment, the invention provides any of the above-describedmethods for the treatment of an HCV infection, where the subject methodis modified to include the steps of (1) identifying a patient having anHCV infection characterized by any of HCV genotypes 5, 6, 7, 8 and 9 andthen (2) administering to the patient the drug therapy of the subjectmethod for a time period of at least about 24 weeks and up to about 48weeks.

Subjects Suitable for Treatment

The subject methods are suitable for treating individuals having, orsusceptible to having, an alphavirus infection, e.g., a flavivirusinfection (e.g., an HCV infection, etc.). The subject methods are alsosuitable for treating individuals who have been previously treated foran alphavirus infection with an agent (other than an α-glucosidaseinhibitor as discussed above) and are refractory to treatment with theagent, and who have either failed the previous treatment; or who cannottolerate treatment with the non-α-glucosidase agent; or who responded tothe previous treatment and relapsed. In many embodiments, the individualis a human.

Individuals who have been clinically diagnosed as infected with analphavirus are suitable for treatment with a method of the instantinvention. Of particular interest in some embodiments are individualswho have been clinically diagnosed as infected with a hepatitis virus(e.g., HAV, HBV, HCV, delta, etc.), particularly HCV. Of particularinterest in other embodiments are individuals who have been clinicallydiagnosed as infected with West Nile Virus.

Individuals who are to be treated according to the methods of theinvention include individuals who have been clinically diagnosed asinfected with HCV. Individuals who are infected with HCV are identifiedas having HCV RNA in their blood, and/or having anti-HCV antibody intheir serum.

Individuals who are clinically diagnosed as infected with HCV includenaïve individuals (e.g., individuals not previously treated for HCV,particularly those who have not previously received IFN-α-based and/orribavirin-based therapy) and individuals who have failed prior treatmentfor HCV (“treatment failure” patients). Treatment failure patientsinclude non-responders (i.e., individuals in whom the HCV titer was notsignificantly or sufficiently reduced by a previous treatment for HCV,e.g., a previous IFN-α monotherapy, a previous IFN-α and ribavirincombination therapy, or a previous pegylated IFN-α and ribavirincombination therapy); and relapsers (i.e., individuals who werepreviously treated for HCV, e.g., who received a previous IFN-αmonotherapy, a previous IFN-α and ribavirin combination therapy, or aprevious pegylated IFN-α and ribavirin combination therapy, whose HCVtiter decreased, and subsequently increased).

In particular embodiments of interest, individuals have an HCV titer ofat least about 10⁵, at least about 5×10⁵, or at least about 10⁶, or atleast about 2×10⁶, genome copies of HCV per milliliter of serum. Thepatient may be infected with any HCV genotype (genotype 1, including 1aand 1b, 2, 3, 4, 6, etc. and subtypes (e.g., 2a, 2b, 3a, etc.)),particularly a difficult to treat genotype such as HCV genotype 1 andparticular HCV subtypes and quasispecies.

Also of interest are HCV-positive individuals (as described above) whoexhibit severe fibrosis or early cirrhosis (non-decompensated,Child's-Pugh class A or less), or more advanced cirrhosis(decompensated, Child's-Pugh class B or C) due to chronic HCV infectionand who are viremic despite prior anti-viral treatment with IFN-α -basedtherapies or who cannot tolerate IFN-α-based therapies, or who have acontraindication to such therapies. In particular embodiments ofinterest, HCV-positive individuals with stage 3 or 4 liver fibrosisaccording to the METAVIR scoring system are suitable for treatment withthe methods of the present invention. In other embodiments, individualssuitable for treatment with the methods of the instant invention arepatients with decompensated cirrhosis with clinical manifestations,including patients with far-advanced liver cirrhosis, including thoseawaiting liver transplantation. In still other embodiments, individualssuitable for treatment with the methods of the instant invention includepatients with milder degrees of fibrosis including those with earlyfibrosis (stages 1 and 2 in the METAVIR, Ludwig, and Scheuer scoringsystems; or stages 1, 2, or 3 in the Ishak scoring system.).

In some embodiments of interest, a subject is an individual who is HCVinfected and who is diabetic, e.g. who has either Type I diabetes orType II diabetes.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 Effect of α-Glucosidase Inhibitors on Viral Replication in Invitro Cell Culture Materials and Methods

Cells and virus. A bovine turbinate (BT) intestinal cell line(ATCC-CRL1390) and Madin-Darby bovine kidney cells (ATCC-CCL22) weregrown in DMEM/F12 (Gibco/BRL, Gaitherburg, Md.) supplemented with 10%heat-inactivated horse serum (Gibco/BRL). A549 cells (ATCC-CCL185) weremaintained in culture medium supplemented 10% fetal bovine serum(Gibco/BRL). BVDV cytopathic strain NADL (ATCC-VR534) and Vesicularstomatitis virus (VSV) Indiana serotype were used in this study. Thevirus stock was plaque purified three times on BT cell monolayers priorto large scale virus stock preparation. The titers of stock solution ofthe virus was determined to be 6×10 ⁵ PFU/ml.

Alpha-Glucosidase inhibitors, and IFM. Castanospermine (CTS)(Calbiochem, La Jolla, Calif.) was prepared as a concentrated stock at100 mM in water. Acarbose (Precose; Bayer) and miglitol (Glycet;Pharmacia) were obtained from commercial sources. Both of CTS andacarbose were prepared in water at 100 mM. Infergen consensus alphainterferon (CIFN) and Actimmune interferon gamma (InterMune, Brisbane,Calif.) were diluted in MDBK media at 10 ng/ml and 100 ng/ml.

BVDV reduction assay. MDBK monolayers (5×10⁴ per well of a 96 wellplate) were infected with BVDV strain NADL at multiplicity of infection(MOI)=0.01 or 0.005. At 1 hour post-infection, the inoculum was removedand the cultures were washed twice in MDBK media. Media containingacarbose, miglitol, CST, or interferons (IFNs) alone or in combinationswere added to the cultures. At 48 hours to 72 hours post-infection, thecultures were harvested and frozen at −70° C. before being used forviral RNA purification.

VSV, BVDV single-cycle replication. A549 or MDBK cells (5×10⁴ per wellof a 96 well plate) were infected in triplicate at a multiplicity ofinfection of >1 or MOI=0.01. At 1 hour post-infection, the inoculum wasremoved and the culture was washed twice with 100 ul of A549 or MDBKmedia. Media containing acarbose, miglitol, CST, or IFNs alone or incombinations were added to the cultures. At 24 hours post-infection, thecultures were harvested and frozen at −70° C. before being used forviral RNA purification.

Viral RNA purification and Real-Time RT-PCR analysis. RNA from releasedviral particles was purified using the QIAamp vial RNA purification kit(Qiagen) with 140 ul of culture supernatant as the stating material.Serial dilutions of VSV RNA or BVDV RNA were prepared from samples ofinfected cell cultures and used as absolute standards for quantitationof viral RNA. Each plaque was assumed to be the result of cell deathcaused by the progeny of one infective virus, thus the virus titer wasexpressed as plaque forming units (PFU) per ml, which is equivalent tocopies per ml. Each sample was measured in triplicate, using the ABIPrism 7900HT Sequence Detection System (Applied Biosystems). The primersused for real-time reverse transcription polymerase chain reaction(RT-PCR) amplification of fragments of BVDV (AJ133738) 5′ non-translatedregion (NTR) were (a) sense 5′-CCATGCCCTTAGTAGGACTAGCA-3′ (SEQ ID NO:1)and (b) antisense 5′-TTCCAAGGCGTCGAACCA-3′ (SEQ ID NO:2) The TaqManprobe was 5′-AACAGTGGTGAGTTCGTTGGATGGCTTAAG-3′ (SEQ ID NO:3) for 5′NTR.The primers used for real-time RT-PCR amplification of fragments of BVDV(AJ133738) NS5B regions were (a) sense 5′-GTTGGAGATTTTCCACACGATAGC-3′(SEQ ID NO:4) and (b) antisense 5′-CCCCGCCTCAAGTTGCT-3′ (SEQ ID NO:5).The TaqMan probe was 5′-CAACCCACCCTGAAACACACCTACGG-3′ (SEQ ID NO:6) forthe NS5B region. TaqMan FAM/TAMRA-labeled hybridization probes weresupplied as mixtures and were used in a 50 μl single tube PCR reaction.The reverse transcription (RT) step was performed at 48° C. for 30 min,followed by 10 min at 95° C., then followed by 40 cycles of 15 secondsat 95° C. and 1 minute at 60° C.

Results and Discussion

The results of the VSV and BVDV single-cycle replication assaysindicated that VSV and BVDV replication is sensitive to glucosidaseinhibitors. In cells infected with VSV at a multiplicity of infection(MOI) >1, none of the glucosidase inhibitors (miglitol, acarbose andpositive control CTS, which prevents the action of glucosidase I and II)exhibited antiviral activity. In VSV-infected cells at MOI=0.01, all ofthe glucosidase inhibitors (miglitol, acarbose and positive control CTS)exhibited antiviral activity at a concentration of 1 mM. Although onlythe positive control CTS exhibited antiviral activity in BVDV-infectedcells at MOI >1, all of the glucosidase inhibitors (miglitol, acarboseand positive control CTS) exhibited antiviral activity in BVDV-infectedcells at MOI=0.05 [v. 0.005] (data shown in FIGS. 1-3).

The results of the BVDV viral reduction assays indicated that bothmiglitol and acarbose are potent antiviral agents that inhibit BVDVreplication.

Example 2 Effect of Combinations of α-glucosidase Inhibitors and IFN-αon Viral Replication in In vitro Cell Culture

The reduction in viral infectivity caused by miglitol is been performedin BVDV, a surrogate model for HCV. MDBK cells were infected by BVDVvirus first, and then were treated by miglitol alone or combined withCIFN. After 48 hrs treatments, the BVDV virus was tested to compare withuntreated cells by real-time PCR. The results are shown in FIGS. 5-12.Table 2 provides the concentrations (in pg/ml CIFN and μM miglitol) ofCIFN and miglitol in the combinations depicted in FIG. 7.

TABLE 2 combo 1 CIFN 450 + miglitol 450 combo 2 CIFN 150 + miglitol 150combo 3 CIFN 50 + miglitol 50 combo 4 CIFN 16.7 + miglitol 16.7 combo 5CIFN 5.6 + miglitol 5.6

The results indicate that miglitol has an ant-viral potential in HCVpatients. The results further indicate that miglitol plus CIFN has asynergistic effect: Combination Index (CI) value in ED50 is 0.86 (slightsynergism), in ED 75 is 0.71 (moderate synergism) and ED 90 is 0.58(synergism), respectively. The data are depicted in Table 3, below.

TABLE 3 Combination Index Values at Drug ED50 ED75 ED90 Dm m r COMBOmean 0.85285 0.6994 0.57605 1.07223 0.32336 0.98042 (1:1) 0.856970.70455 0.58249 Mutually non- exclusive CIFN N/A N/A N/A 1.26443 0.30510.96475 miglitol N/A N/A N/A 220.7982   0.36987 0.95355

Example 3 Effect of Combinations of α-Glucosidase Inhibitors and/orIFN-α and/or an HCV NS3 Protease Inhibitor on Viral Replication in Invitro Cell Culture

The reduction in viral infectivity caused by miglitol is been performedin BVDV, a surrogate model for HCV. MDBK cells were infected by BVDVvirus first, and then were treated by miglitol alone or combined withCIFN. After 48 hrs treatments:the BVDV virus was tested to compare withuntreated cells by real-time PCR. The results are shown in Tables 4-10.

TABLE 4 CIFN BVDV BVDV Mean BVDV Standard (pg/ml) vRNA copy vRNA copyvRNA copy IC50 Deviation 64 133,283.16 66,744.57 100,013.865 80.233,269.3 32 177,943.2 117,714.05 147,828.625 70.7 30,114.6 16 193,559.75181,262.64 187,411.195 62.9 6,148.6 8 227,865.22 279,692.53 253,778.87549.7 25,913.7 4 242,459.25 328,102.16 285,280.705 43.5 42,821.5 2283,995.77 294,840.88 289,418.325 42.6 5,422.6 1 303,367 323,186.25313,276.625 37.9 9,909.6 0.5 367,344.5 381,928.16 374,636.33 25.77,291.8 0.25 425,421 414,415.48 419,918.24 16.8 5,502.8 0.125 439,664.17460,441.38 450,052.775 10.8 10,388.6 Mock 504,483.08

TABLE 5 miglitol BVDV BVDV Mean BVDV Standard (uM) vRNA vRNA copy vRNAcopy IC50 Deviation 800 81,020.45 108,846.34 94,933.395 82.3 13,912.9400 309,639.16 246,917.33 278,278.245 48.3 31,360.9 200 325,059.84270,873.97 297,966.905 44.6 27,092.9 100 429,782.12 173,197.69301,489.905 43.9 128,292.2 50 306,240.62 300,179.25 303,209.935 43.63,030.7 25 397,297.53 287,921.75 342,609.64 36.3 54,687.9 12.5404,207.16 479,795.72 442,001.44 17.8 37,794.3 6.25 546,161.3 347,250.03446,705.665 16.9 99,455.6 3.125 468,509.28 641,705.5 555,107.39 −3.286,598.1 1.5625 550,621.8 591,071.3 −570,846.55 −6.1 20,224.8 Mock537,859.66

TABLE 6 NS3 BVDV BVDV Mean BVDV Standard (nM) vRNA copy vRNA copy vRNACopy IC50 Devation 200.00 25,201.072 15,755.012 20,478.042 89.2 4,723.0100.00 51,336.78 42,620.09 46,978.435 72.6 4,358.3 50.00 67,876.947,705.5 57,791.2 66.2 10,085.7 25.00 70,181.76 69,093.43 69,637.59559.3 544.2 12.50 101,798.64 116,275.625 109,037.1445 36.3 7,238.5 6.25130,837.18 110,579.4 120,708.29 29.5 10,128.9 3.13 137,392.88111,570.555 124,481.7175 27.3 12,911.2 1.56 137,386.48 122,571.445129,978.9625 24.1 7,407.5 0.78 171,179.22 112,177.94 141,678.58 17.229,500.6 0.39 181,504.39 113,578.914 147,541.652 13.8 33,962.7 Mock190,022.88

TABLE 7 CIFN (pg/ml) + miglitol (uM) BVDV BVDV Mean BVDV Standard(1:12.5) vRNA copy vRNA copy vRNA Copy IC50 Devation 64 72,456.0291,520.305 81,988.1625 86.4 9,532.1 32 137,889.88 65,299 101,594.44 83.236,295.4 16 216,909.62 121,311.625 169,110.6225 72.0 47,799.0 8174,092.38 214,850.2 194,471.29 67.8 20,378.9 4 192,806.98 209,206.38201,006.68 66.7 8,199.7 2 255,973.94 347,635.75 301,804.845 50.045,830.9 1 143,606.42 507,253.1 325,429.76 46.1 181,823.3 0.5 642,918.44366,290.84 504,604.64 16.5 138,313.8 0.25 492,284.75 552,460.44522,372.595 13.5 30,087.8 0.125 786,990.3 581,912.25 684,451.275 −13.3102,539.0 Mock 603,964.25

TABLE 8 NS3 (nM) + miglitol (uM) BVDV BVDV Mean BVDV Standard (1:4) vRNAcopy vRNA copy vRNA Copy IC50 Devation 200.00 9,903.573 9,519.90459,711.73875 96.6 191.8 100.00 96,224.62 10,155.769 53,190.1945 81.543,034.4 50.00 105,968.4 88,732.51 97,350.45 66.2 8,617.9 25.0096,991.73 104,053.125 100,522.4275 65.1 3,530.7 12.50 101,847.5114,010.137 107,928.8085 62.5 6,081.3 6.25 132,863.1 124,937.25128,900.155 55.3 3,962.9 3.13 144,261.5 137,993.336 141,127.418 51.03,134.1 1.56 181,269.3 172,079.26 176,674.285 38.7 4,595.0 0.78204,120.6 164,474.562 184,297.591 36.0 19,823.0 0.39 203,088 204,666.99203,877.495 29.2 789.5 Mock 288,147.44

TABLE 9 CIFN (pg/ml) + miglitol (uM) + NS3 (nM) BVDV BVDV Mean BVDVStandard (1:3.125:12.5) vRNA copy vRNA copy vRNA Copy IC50 Devation 64878.925 790.69156 834.80828 99.7 44.1 32 18,100.62 18,440.325718,270.47285 93.5 169.9 16 37,153.125 35,230.028 36,191.5765 87.1 961.58 44,393.77 41,493.951 42,943.8605 84.7 1,449.9 4 59,090.88 58,344.7558,717.815 79.1 373.1 2 135,737.77 123,298.062 129,517.916 53.9 6,219.91 135,542.05 138,580.41 137,061.23 51.3 1,519.2 0.5 157,368.77132,176.69 144,772.73 48.5 12,596.0 0.25 180,111.42 174,488.363177,299.8915 37.0 2,811.5 0.125 194,642.1 193,841.48 194,241.79 30.9400.3 Mock 281,254.88

The results demonstrate that miglitol plus CIFN and/or an NS3 proteaseinhibitors have synergistic effects. The results showed thatCIFN+miglitol, the Combination Index Values (CI) at ED90 is 0.31(indicating strong synergism); in HCV NS3/4 protease inhibitor+miglitol,the CI values at ED90 is 0.40 (indicating synergism); and inCIFN+miglitol+NS3/4 protease inhibitor, the CI values at ED90 is 0.077(indicating very strong synergism). The combination resulted in a 4.8fold decrease in the EC90 of CIFN combined with miglitol and 48 folddecreases in the EC90 of CIFN combined with miglitol and HCV NS3/4protease inhibitor together.

TABLE 10 Dosage @ CI @ Dosage @ CI @ Dosage @ CI @ ED50 ED50 ED75 ED75ED90 ED90 CIFN (pg/ml) + 2.72 pg/ml + 0.76 14.79 pg/ml + 0.48 80.35pg/ml + 0.31 miglitol (uM) 34.04 uM 184.91 uM 1004.37 uM (1:12.5) NS3(nM) + 3.41 nM + 0.33 26.22 nM + 0.36 201.52 pg/ml + 0.40 miglitol (uM)13.64 uM 104.86 uM 806.07 uM (1:4) CIFN (pg/ml) + 0.68 pg/ml + 0.34 2.44pg/ml + 0.16 8.82 pg/ml + 0.07 NS3 (nM) + 2.11 nM + 7.63 nM + 27.57 nM +miglitol (uM) 8.46 uM 30.53 uM 110.28 uM (1:3.125:12.5)

Example 4 Methods for Treating HCV with the Addition of NS5b RdRpInhibitors

The present invention provides methods for testing the effectiveness ofco-treating with alpha-glucosidase and NS5b RdRp inhibitors.

Materials and Methods

Cells and virus. A bovine turbinate (BT) intestinal cell line(ATCC-CRL1390) and Madin-Darby bovine kidney cells (ATCC-CC L22) aregrown in DMEM/F12 (Gibco/BRL, Gaitherburg, Md.) supplemented with10%heat-inactivated horse serum (Gibco/BRL). A549 cells (ATCC-CCL185) aremaintained in culture medium supplemented with 10% fetal bovine serum(Gibco/BRL). BVDV cytopathic strain NADL (ATCC-VR534) and Vesicularstomatitis virus (VSV) Indiana serotype are used in this study. Thevirus stock is plaque purified three times on BT cell monolayers priorto large scale virus stock preparation. The titers of stock solution ofthe virus is determined to be 6×10 ⁵ PFU/ml.

Alpha-Glucosidase inhibitors, IFN and HCV NS5b RdRp inhibitors.Castanospermine (CTS) (Calbiochem, La Jolla, Calif.) is prepared as aconcentrated stock at 100 mM in water. CTS and acarbose are prepared inwater at 100 mM. Infergen consensus alpha interferon (CIFN) andActimmune interferon gamma (Intermune, Brisbane, Calif.) are diluted inMDBK media at 10 ng/ml and 100 ng/ml. Two NS5b RdRp inhibitors:2′-O-Me-C (IC50 20 mM in replicon JBC 2003, p 11979) and 2′-F-C (EC90 5mM in replicon, Antimicrobal Agents and Chemother, 2004, p 651) are alsoused.

BVDV reduction assay. MDBK monolayers (5×10⁴ per well of a 96 wellplate) are infected with BVDV strain NADL at multiplicity of infection(MOI)=0.01 or 0.005. At 1 hour post-infection, the inoculum is removedand the cultures are washed twice in MDBK media. Media containingacarbose, miglitol, CST, interferons (IFNs) or NS5b RdRp inhibitorsalone or in combination are added to the cultures. At 48 hours to 72hours post-infection, the cultures are harvested and frozen at −70° C.before being used for viral RNA purification.

VSV, BVDV single-cycle replication. A549 or MDBK cells (5×10⁴ per wellof a 96 well plate) are infected in triplicate at a multiplicity ofinfection of >1 or MOI=0.01. At 1 hour post-infection, the inoculum isremoved and the culture is washed twice with 100 ul of A549 or MDBKmedia. Media containing acarbose, miglitol, CST, IFNs or NS5b RdRpinhibitors alone or in combination are added to the cultures. At 24hours post-infection, the cultures are harvested and frozen at −70° C.before being used for viral RNA purification.

Viral RNA purification and Real-Time RT-PCR analysis. RNA from releasedviral particles is purified using the QIAamp vial RNA purification kit(Qiagen) with 140 ul of culture supernatant as the starting material.Serial dilutions of VSV RNA or BVDV RNA are prepared from samples ofinfected cell cultures and used as absolute standards for quantizationof viral RNA. Each plaque is assumed to be the result of cell deathcaused by the progeny of one infective virus, thus the virus titer isexpressed as plaque forming units (PFU) per ml, which is equivalent tocopies per ml. Each sample is measured in triplicate, using the ABIPrism 7900HT Sequence Detection System (Applied Biosystems). The primersto be used for real-time reverse transcription polymerase chain reaction(RT-PCR) amplification of fragments of BVDV (AJ133738) 5′ non-translatedregion (NTR) are (a) sense 5′-CCATGCCCTTAGTAGGACTAGCA-3′ (SEQ ID NO:1)and (b) antisense 5′-TTCCAAGGCGTCGAACCA-3′ (SEQ ID NO:2). The TaqManprobe is 5′-AACAGTGGTGAGTTCGTTGGATGGCTTAAG-3′ (SEQ ID NO:3) for 5′NTR.The primers to be used for real-time RT-PCR amplification of fragmentsof BVDV (AJ133738) NS5B regions are (a) sense5′-GTTGGAGATTTTCCACACGATAGC-3′ (SEQ ID NO:4) and (b) antisense5′-CCCCGCCTCAAGTTGCT-3′ (SEQ ID NO:5). The TaqMan probe is5′-CAACCCACCCTGAAACACACCTACGG-3′ (SEQ ID NO:6) for the NS5B region.TaqMan FAM/TAMRA-labeled hybridization probes are supplied as mixturesand are to be used in a 50 μl single tube PCR reaction. The reversetranscription (RT) step is to be performed at 48° C. for 30 min,followed by 10 min at 95° C., then followed by 40 cycles of 15 secondsat 95° C. and 1 minute at 60° C.

Results and Discussion

After the above treatments, the results will indicate whether VSV andBVDV replication is sensitive or not sensitive to NS5b RdRp inhibitors.The cells may be sensitive at a MOI>1, at a MOI>0.01, at a MOI≦0.01, orat a MOI=0.05. Sensitivity is determined by the presence of antiviralactivity, the determination of which is well known in the art.

In one embodiment, NS5b RdRp inhibitors exhibit antiviral activity.

In another embodiment, alpha-glucosidase and NS5b RdRp inhibitors areeffective to produce a reduction in viral infectivity.

In another embodiment, alpha-glucosidase and NS5b RdRp inhibitors areeffective antiviral agents in combination to inhibit BVDV replication.

In another embodiment, the combination effect of alpha-glucosidase andNS5b RdRp inhibitors is greater than the effect of eitheralpha-glucosidase or NS5b RdRp inhibitors alone.

In another embodiment, an effective amount of alpha-glucosidaseinhibitor and NS5b RdRp inhibitor is an amount that is effective toreduce the viral load in a patient.

In one embodiment, an effective amount of alpha-glucosidase inhibitorand NS5b RdRp inhibitor is an amount that is effective to achieve SVR ina patient.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of treating a flavivirus infection in an individual, themethod comprising administering to the individual an effective amount ofan agent that inhibits enzymatic activity of a membrane-boundα-glucosidase.
 2. The method of claim 1, wherein the agent inhibits thep7 protein of hepatitis C virus.
 3. The method of claim 1, wherein theagent is an imino sugar.
 4. The method of claim 1, wherein the agent isacarbose.
 5. The method of claim 1, wherein the agent is miglitol.
 6. Amethod of treating a flavivirus infection in an individual, the methodcomprising administering to the individual effective amounts of anα-glucosidase inhibitor and at least one additional therapeutic agent.7. The method of claim 6, wherein the at least one additionaltherapeutic agent comprises an interferon-α (IFN-α).
 8. The method ofclaim 7, wherein the IFN-α is interferon alfacon-1.
 9. The method ofclaim 8, wherein the IFN-α is pegylated.
 10. The method of claim 9,wherein the pegylated IFN-α is selected from peginterferon alfa-2a,peginterferon alfa-2b, and monoPEG (30 kD, linear)-ylated consensusIFN-α.
 11. The method of claim 8, wherein the IFN-α ishyperglycosylated.
 12. The method of claim 6, wherein the at least oneadditional therapeutic agent comprises an interferon-γ (IFN-γ).
 13. Themethod of claim 12, wherein the IFN-γ is interferon gamma-1b.
 14. Themethod of claim 13, wherein the IFN-γ is pegylated.
 15. The method ofclaim 13, wherein the IFN-γ is hyperglycosylated.
 16. The method ofclaim 6, wherein the at least one additional therapeutic agent comprisesan HCV NS3 protease inhibitor.
 17. The method of claim 6, wherein the atleast one additional therapeutic agent comprises an HCV NS5BRNA-dependent RNA polymerase inhibitor.
 18. The method of claim 6,wherein the at least one additional therapeutic agent comprises anucleoside analog.
 19. The method of claim 18, wherein the nucleosideanalog is selected from viramidine, ribavirin, and levovirin.
 20. Themethod of any one of claims 1-19, wherein the individual is a human.